bluuebunny/crossref_metadata_2025_split huggingface dataset:
Different isoforms of human pituitary thyroid-stimulating hormone have different relative biological activities
Authors: Pickles A. J. , Peers N. , Robertson W. R. , Lambert A. 
Published: December 2008

ABSTRACT
The relationship between the immunological and biological activities of thyroid-stimulating hormone (TSH) isoforms present in the three human pituitary preparations 68/38 (1st IRP), 80/558 (2nd IRP) and 63/14 (MRC Research Standard A) was investigated. The isoforms were separated by chromatofocusing. Six peaks of immunoactivity were detected in 80/558, with pI values (means±s.e.m.) of 6·6±0·1, 6·2±0·1, 5·9±0·1, 5·5±0·1, 5·2±0·1 and 4·9±0·1. Four peaks, with pi values of 6·8±0·1, 5·9±0·1, 5·5±0·1 and 5·2±0·1, were observed for 68/38. Standard 63/14 had five peaks, with pI values of 6·9±0·1, 6·4±0·1, 5·9±0·1, 5·4±0·1 and 4·9±0·1. For each standard, six fractions around the peak areas and at the top and bottom of the gradient were pooled and microconcentrated to <1·0ml. Microconcentrated TSH samples were assayed in three TSH bioassays based upon FRTL-5 thyroid cells, utilizing cyclic AMP accumulation, iodide and thymidine uptake as end-points and standard 80/558 as reference preparation. The more acidic forms of TSH showed a higher biological: immunological (B: I) ratio for cyclic AMP accumulation with, for example, 63/14 having a maximum of 3·7 (pI 4·9) and a minimum of <0·7 (pI 6·9). In contrast, the maximum and minimum B: I ratios for iodide uptake for 63/14 were 3·8 (pI 6·9) and <0·8 (pI 4·6), and for thymidine uptake, maximum and minimum ratios were 7·2 (pI 6·9) and 1·1 (pI 4·6) respectively. In conclusion, the acidic forms of TSH stimulated cyclic AMP accumulation more than the alkaline forms, whereas the opposite was shown for iodide uptake and thymidine uptake.
Characterization of rat gastric inhibitory peptide cDNA
Authors: Sharma S. K. , Austin C. , Howard A. , Lo G. , Nicholl C. G. , Legon S. 
Published: December 2008

ABSTRACT
Gastric inhibitory peptide (GIP) is a 42 amino acid gastrointestinal peptide which inhibits gastric acid secretion and stimulates pancreatic insulin secretion in the presence of glucose. Here we report the sequence of the cDNA encoding the rat GIP precursor. PreproGIP was 144 amino acids in length and comprised the GIP peptide itself, N- and C-terminal flanking peptides of 22 and 59 amino acids respectively and a typical hydrophobic signal peptide. The sequence indicated that GIP is released from its precursor by cleavage at single arginine residues. The C-terminal flanking peptide may have an important function since it was well conserved and contained a region of 16 amino acids with only a single, conservative replacement. Rat GIP mRNA was found in the duodenum and jejunum. Levels of GIP mRNA in the duodenum were increased twofold after a period of 2 days of starvation. There was no detectable expression of the GIP gene in other parts of the gastrointestinal tract or in other endocrine tissues. However, in pancreatic mRNA preparations, a larger mRNA was detected after low stringency hybridization. This could represent a further member of this gene family.
Glucocorticoid-mediated responses of plasma ACTH and anterior pituitary pro-opiomelanocortin, growth hormone and prolactin mRNAs during adjuvant-induced arthritis in the rat
Authors: Stephanou A. , Sarlis N. J. , Knight R. A. , Lightman S. L. , Chowdrey H. S. 
Published: December 2008

ABSTRACT
Adjuvant arthritis (AA) in the rat leads to chronic stimulation of the hypothalamic-pituitary-adrenal (HPA) axis and the loss of its diurnal rhythmicity. We have investigated the effects of adrenalectomy (ADX) and different levels of corticosterone replacement upon plasma ACTH levels and anterior pituitary pro-opiomelanocortin (POMC), GH and prolactin mRNAs during the development of AA. In control ADX animals, we observed the negative feedback effects of exogenous corticosterone on plasma ACTH and anterior pituitary POMC mRNA. In the ADX animal with AA, however, the increased POMC mRNA which was observed was not reduced by exogenous corticosterone on day 7 of AA, although the negative feedback effect of corticosterone on plasma ACTH was intact. On day 14, however, even high dose corticosterone replacement failed to have a significant feedback effect on the raised levels of plasma ACTH.
In control ADX animals, corticosterone replacement resulted in increased anterior pituitary GH mRNA and reduced prolactin mRNA. In contrast, in ADX animals with AA, GH mRNA was reduced and there was a further decrease in prolactin mRNA. In these animals, corticosterone replacement did not affect GH or prolactin mRNA expression.
These data demonstrate a disruption of the normal mechanisms underlying feedback inhibition of the HPA axis by glucocorticoids during AA. Similarly, the glucocorticoid-dependent regulation of GH and prolactin mRNA expression is altered in AA.
Cloning of a bullfrog growth hormone cDNA: expression of growth hormone mRNA in larval and adult bullfrog pituitaries
Authors: Takahashi N. , Kikuyama S. , Gen K. , Maruyama O. , Kato Y. 
Published: December 2008

ABSTRACT
A GH cDNA was specifically amplified from cDNAs constructed from total RNA of bullfrog (Rana catesbeiana) adenohypophyses employing the DNA polymerase chain reaction. Sequencing analysis revealed that the cDNA clone thus obtained was 654 bp in length, and included an open reading frame encoding the entire sequence of mature GH, with its signal peptide. Slight discrepancies were noted between the deduced amino acid sequence and that determined by direct protein sequencing of purified bullfrog GH or that deduced from the nucleotide sequence reported previously. The length of the bullfrog GH mRNA was estimated to be about 1·2 kb by Northern blot analysis. Homologies of nucleotide and amino acid sequences between GH and prolactin of bullfrog origin were 48% and 26% respectively. Using the cDNA as a probe, the content of GH mRNA in the pituitary of larval and adult bullfrogs was measured. GH mRNA levels were relatively low at the preclimax stage, and rose markedly during climax. In juvenile frogs, GH mRNA levels in the pituitary were extremely high and declined towards adulthood. This finding suggests that the increase in plasma and pituitary GH levels reported previously accompanies the increase in GH synthesis.
Detection of retinoic acid receptor mRNA in rat tissues by reverse transcriptase-polymerase chain reaction
Authors: Wan Y.-J. Y. , Wang L. , Wu T.-C. J. 
Published: December 2008

ABSTRACT
The presence of retinoic acid receptor (RAR) α, β and γ mRNA was examined in 16 different kinds of rat tissue using the highly sensitive reverse transcriptase-polymerase chain reaction technique. The data demonstrated that each tissue expressed at least two types of RAR mRNA. Among the three types of RAR mRNA, RAR α was widely expressed in all types of organ and was the dominant form expressed in the gastrointestinal tract. RAR β mRNA was not present in the intestine and spleen. In addition, RAR β mRNA levels were high in the heart, lung, brain, testis and epididymis. RAR γ mRNA was abundant in both male and female reproductive systems, as well as epidermal tissues. The prevalence of each RAR mRNA in the tissues suggests the diverse biological roles of these receptors.
Glucocorticoid inhibition of ACTH peptides: small cell lung cancer cell lines are more resistant than pituitary corticotroph adenoma cells
Authors: Farrell W E , Stewart M F , Clark A J L , Crosby S R , Davis J R E , White A 
Published: December 2008

ABSTRACT
In the normal pituitary, glucocorticoids are the principal negative regulator of the pro-opiomelanocortin (POMC) gene which gives rise to the biologically active peptides ACTH and β-endorphin. In Cushing's syndrome, ACTH-secreting pituitary tumours show a degree of glucocorticoid resistance, whilst ACTH-secreting extra-pituitary tumours have an even greater resistance to glucocorticoid excess. In an attempt to understand the mechanism of this phenomenon, we have compared the effects of glucocorticoids on POMC mRNA and peptide secretion in human and mouse corticotroph adenoma cells and in small cell lung carcinoma (SCLC) cells. ACTH precursor peptides were inhibited within 24 h by 25–50 nm hydrocortisone in primary cultures from a human corticotroph adenoma. In the mouse corticotroph adenoma cell line (AtT20), inhibition of both ACTH precursors and ACTH was not observed after 24 h but, by 10 days, glucocorticoids suppressed peptide levels with a concentration causing 50% inhibition of 50 nm hydrocortisone and maximal inhibition at 500 nm hydrocortisone. In marked contrast, there was no response to 500 nm hydrocortisone in the five SCLC cell lines (COR L103, COR L42, COR L24, COR L31, DMS 79) all of which secrete ACTH precursors. However, two of the five SCLC cell lines (COR L31 and DMS 79) were responsive to 1000 nm hydrocortisone. POMC mRNA, quantitated by slot-blot analysis, gave similar results for the five SCLC cell lines, implying that the abnormality may occur at the level of gene expression. When one of the three resistant cell lines (COR L103) was incubated with 2000 nm hydrocortisone or 2000 nm dexamethasone a clear suppression of precursor peptides and POMC mRNA was observed. This suggests that the resistance to glucocorticoid inhibition is relative rather than absolute, implying that the normal mechanism is functioning but impaired. Furthermore, there is at least a 20-fold difference in the responsiveness to glucocorticoid inhibition between pituitary and extra-pituitary tumour cells in vitro, which may signify a difference in the underlying mechanism in these two cell types.
Post-transcriptional regulation of bovine parathyroid hormone synthesis
Authors: Hawa N S , O'Riordan J L H , Farrow S M 
Published: December 2008

ABSTRACT
Incubation of bovine parathyroid cells for 48 h in 0·4 mmol calcium/l had no significant effect on steady-state preproparathyroid hormone (preproPTH) mRNA levels when compared with cells incubated in 1·0 mmol calcium/l, but low calcium concentrations increased the membrane-bound polysomal content of preproPTH mRNA by 200±16% (mean±s.d.). No preproPTH mRNA was detected on free polysomes. Actinomycin D (5 and 10 μg/ml) had no effect on steady-state preproPTH mRNA levels measured in dot-blot assays after 24 h, but reduced levels in cells incubated in 1·0 mmol calcium/l to 54±16% and 39±12% of control values respectively after 48 h of incubation. Similarly, in cells incubated in 0·4 mmol calcium/l, actinomycin D (5 and 10μg/ml) reduced steady-state preproPTH mRNA levels to 57±13% and 45±5% of control values respectively. Actinomycin D did not prevent the rise in polysomal content of preproPTH mRNA induced in cells by incubation in 0·4 mmol calcium/l, but increased polysomal content in cells incubated in 0·4 and 1·0mmol calcium/l by 159±9% and 164±13% respectively after 48 h. These results demonstrate post-transcriptional regulation of PTH synthesis in cultured bovine parathyroid cells, and suggest that this control involves a protein which may be calcium-sensitive.
Interaction of diazoxide and cromakalim with ATP-regulated K+ channels in rodent and clonal insulin-secreting cells
Authors: Jaggar J H , Harding E A , Ayton B J , Dunne M J 
Published: December 2008

ABSTRACT
The hyperglycaemia-inducing sulphonamide diazoxide has been previously shown to mediate its effects upon insulin secretion by opening K+ channels and hyperpolarizing the β-cell membrane. The target site has been characterized as the ATP-regulated K+ (K+ATP) channel protein. In the present study, a detailed investigation of interactions of diazoxide and another K+ channel opener, cromakalim, with K+ATP channels has been performed in individual insulin-secreting cells using patchclamp techniques. In agreement with previous studies, diazoxide and cromakalim were found to be effective only when ATP was present upon the inside face of the plasma membrane. The ability of both diazoxide and cromakalim to open channels was, however, found to diminish with time following isolation of inside-out patches. Within seconds of forming the recording configuration, the actions of both compounds were potent, and were found to decline steadily as the number of operational channels decreased ('run-down'). In open cells, where the plasma membrane remains partially intact, the rate of run-down was significantly reduced, and effects of channel openers were recorded up to 80 min following cell permeabilization. We also demonstrated that in the absence of ATP, but in the presence of ADP, both diazoxide and cromakalim were able to open K+ATP channels. Interestingly, once the effects of diazoxide and cromakalim on K+ATP channels in the presence of ATP were lost, both compounds opened channels in the presence of ADP. One implication of these data is that the actions of diazoxide and cromakalim involve regulatory proteins associated with the ion channel; this molecule is able to bind ATP, ADP and possibly other cytosolic nucleotides.
The thyrotrophin hormone receptor of Graves' disease: overexpression of the extracellular domain in insect cells using recombinant baculovirus, immunoaffinity purification and analysis of autoantibody binding
Authors: Huang G C , Page M J , Nicholson L B , Collison K S , McGregor A M , Banga J P 
Published: December 2008

ABSTRACT
Since the cloning of the TSH receptor (TSH-R), the target autoantigen of Graves' disease, the receptor has been expressed in a variety of eukaryotic cells to obtain a functional molecule. Despite this success, the levels of receptor expression have been marginally higher than the extremely low levels found in thyroid cells, preventing any progress on the purification of the molecule. In this study, the large extracellular region of the TSH-R, without the membrane spanning segments, has been expressed in insect cells using recombinant baculovirus to generate substantial quantities of the receptor protein. A monoclonal antibody previously generated to a bacterial TSH-R fusion protein was used to characterize and monitor the expression of the truncated receptor in insect cells. Two polypeptides of 63 and 49 kDa were recognized as the components of the truncated recombinant receptor. The 63 kDa protein was shown to be the glycosylated form of the smaller, 49 kDa, component. Expression in different insect cell lines showed that an increase in expression of approximately tenfold was apparent in High Five cells when compared with Sf21 cells. Very small quantities of the truncated receptor were secreted by the three insect cell lines examined, with the majority of the molecule being retained within the cells. Immunoaffinity purification of milligram quantities of the truncated receptor was achieved using the monoclonal antibody. The availability of the purified TSH-R has allowed the establishment of an enzymelinked immunosorbent assay to measure autoantibodies in the sera of patients with Graves' disease. Although the truncated receptor interacts with autoantibodies, our results show that it does not bind TSH and differs in this respect from other glycoprotein hormone receptors.
Binding specificity and signal transduction of receptors for glucagon-like peptide-1(7–36)amide and gastric inhibitory polypeptide on RINm5F insulinoma cells
Authors: Gallwitz B , Witt M , Fölsch U R , Creutzfeldt W , Schmidt W E 
Published: December 2008

ABSTRACT
Glucagon-like peptide-1(7–36)amide (GLP-1(7–36) amide) and gastric inhibitory polypeptide (GIP), peptides of the glucagon family, stimulate insulin secretion in vitro and in vivo. They possess high N-terminal sequence homology. Binding studies with 125I-labelled GIP and 125I-labelled GLP-1(7– 36)amide were performed in RINm5F insulinoma cells to investigate receptor specificity and to compare both receptors directly. Both binding sites were highly ligand-specific: GIP did not bind to the GLP-1(7–36)amide receptor and vice versa. Both peptides increased intracellular cyclic AMP levels; GLP-1(7– 36)amide was 100-fold more potent in stimulating cyclic AMP production when compared with GIP. At ranges of 1–10 nmol GLP-1(7–36)amide/1 and 0·1–10 GIP/1, corresponding to submaximal binding concentrations, the hormones showed an additive effect on cyclic AMP production. The N-terminal portion of GIP was important for binding, as GIP(1–30) showed almost full binding and biological activity. GIP(17–42) bound in a concentration-dependent manner with approximately 500-fold lower potency than GIP. At concentrations of up to 10 μmol GIP(17–42)/1 no stimulation of cyclic AMP was observed.
Localization of DNase I hypersensitive sites in the human oestrogen receptor gene correlates with the transcriptional activity of two differentially used promoters
Authors: Grandien K F H , Berkenstam A , Nilsson S , Gustafsson J-Å 
Published: December 2008

ABSTRACT
The human oestrogen receptor (hER) gene has recently been shown to be transcribed from two different promoters. In order to study the hER gene promoter region we initially isolated and sequenced 3·3 kb of the 5′ region of the hER gene. DNase I hypersensitivity experiments with nuclei from the ER-expressing cell line MCF-7 mapped three different hypersensitive sites, located in the vicinity of the two hER promoters previously identified. However, in another ER-positive cell line, ZR-75-1, DNase I hypersensitivity was observed only in the region of the downstream promoter, suggesting differences between the two cell lines in promoter usage. Polymerase chain reaction-mediated detection of mRNAs transcribed from the two different promoters demonstrated transcripts from the downstream promoter in both the cell lines studied. However, mRNAs transcribed from the upstream promoter could only be detected in MCF-7 cells. Thus, the pattern of DNase I hypersensitivity correlates with the transcriptional activity of the two promoters in the hER gene. Based on these data, we suggest that differential promoter usage might be a novel level of regulation of the hER gene.
Characterization of the chicken preprogonadotrophinreleasing hormone-I gene
Authors: Dunn I C , Chen Y , Hook C , Sharp P J , Sang H M 
Published: December 2008

ABSTRACT
Partial cDNA clones for chicken gonadotrophin-releasing hormone (GnRH)-I were isolated by reverse transcription-polymerase chain reaction using total RNA from the hypothalami of domestic chickens. Primers for amplification were based on the nucleotide sequence of the mammalian GnRH genes. These amplified clones were used to screen a genomic library from which a series of overlapping clones was isolated. A 63 kb EcoRI fragment containing all the exons and 3·0 kb of the 5′ upstream region was sequenced. The exon—intron structure of the gene was found to be of a similar configuration to those of the mammalian and osteichthyes GnRH genes analysed so far. Individual domains of the predicted prepropeptide are similar to those of mammalian GnRH prepropeptides, comprising a 23 amino acid signal peptide, the decapeptide hormone and a Gly-Lys-Arg cleavage site, followed by a 56 amino acid GnRH-associated peptide. The nucleotide sequence coding for the decapeptide hormone translates into the amino sequence for chicken GnRH-I. The prepropeptide has approximately 50% identity with mammalian prepropeptides and 25% identity with the teleost prepropeptides.
Endotoxin-stimulated production of rat hypothalamic interleukin-1β in vivo and in vitro, measured by specific immunoradiometric assay
Authors: Hagan P , Poole S , Bristow A F 
Published: December 2008

ABSTRACT
Regulation of a number of aspects of the acute-phase response, including induction of fever and activation of the hypothalamo-pituitary-adrenal axis, occurs within the hypothalamus. The acute-phase response appears to be co-ordinated by the inflammatory cytokine interleukin-1 (IL-1). A number of studies using hybridization techniques to measure IL-1 gene expression and immunocyto-chemistry to localize immunoactive IL-1 have established the concept that the central nervous system, and in particular the hypothalamus, is a site of IL-1 production, and that levels increase in response to inflammatory stimuli. In this report we present data on the levels of IL-1β produced in the rat hypothalamus using quantitative immunoassay techniques. Bacterial endotoxin, administered to rats in vivo, evoked increases in hypothalamic IL-1β levels which were significant within 1 h, and reached maximum levels at 5–10 h. The response to endotoxin was dose-related, and levels reached in hypothalamic extracts corresponded to intra-hypothalamic levels of the order of 20 ng/ml. During short-term in-vitro culture of rat hypothalami, endotoxin stimulated a dose-related increase in both the synthesis and the secretion of IL-1β, which reached similar levels to those seen after in-vivo stimulation. Hypothalami obtained from animals stimulated with endotoxin in vivo did not, however, show any evidence of persistent stimulation of IL-1β production when subsequently cultured in vitro.
These data support the concept that production of hypothalamic IL-1 is an essential step in regulating the activity of the hypothalamus during the acute-phase response, and provide for the first time quantitative data on the magnitude, dose—response relationships and time-courses of rat hypothalamic IL-1β production in vivo and in vitro.
Construction of an ecotropic retroviral vector expressing human insulin-like growth factor-I
Authors: McClure F , Dawson T , Wynford-Thomas D 
Published: December 2008

ABSTRACT
There is increasing evidence that IGF-I plays an autocrine role in a wide range of human tumours, including, in particular, adenomas of the thyroid epithelium. To investigate this further, we set out to generate a retrovirus vector which would permit experimental manipulation of the expression of IGF-I in normal and neoplastic epithelial cells. We describe here the construction and validation of a high-titre ecotropic vector which transduces stable expression and secretion of human IGF-IA, as shown by analysis of mRNA and conditioned medium from rodent epithelial target cells. This vector should be a useful tool for assessing the contribution of abnormal IGF-I expression to the neoplastic phenotype.
Cytochrome P-450 17α-hydroxylase protein and mRNA in the testis of the testicular feminized (Tfm) mouse
Authors: O'Shaughnessy P J , Murphy L 
Published: December 2008

ABSTRACT
The testicular feminized (Tfm) mouse lacks functional androgen receptors and develops with a female external phenotype and internal testes. The testes of these animals contain normal, or close to normal, numbers of Leydig cells but secrete very low amounts of androgen due to a lack of 17α-hydroxylase activity. To determine whether this loss of activity is due to a lack of enzyme synthesis or a change in catalytic activity we have examined 17α-hydroxylase cytochrome P-450 (P-45017α) protein and mRNA levels in the testes of Tfm mice.
Levels of P-45017α protein were measured by immunoblotting, while mRNA was measured following reverse transcription (RT) and amplification by the polymerase chain reaction (PCR). Conditions for RT-PCR were determined which allowed semiquantification of P-45017α mRNA relative to β-actin mRNA. In extracts of Tfm testes P-45017α protein was undetectable using antiserum against porcine P-45017α. In contrast, a protein of around 54 kDa was clearly detectable in extracts of control cryptorchid testes. Using RT-PCR, P-45017α mRNA was detectable in both control and [ill] testes but, expressed in terms of β-actin mRNA, levels of P-45017α mRNA in control testes were 40-fold higher than those in [ill] testes. If the total amount of RNA extracted from each testis is taken into account then P-45017α mRNA levels per testis were up to 400-fold higher in control testes. These results show that the reduced level of 17α-hydroxylase activity in [ill] testes is related to reduced protein synthesis. Previous results have shown that androgens reduce P-45017α mRNA levels in cultured Leydig cells. Results from this study suggest, however, that androgens are required to induce normal levels of P-45017α mRNA in Leydig cells.