bluuebunny/crossref_metadata_2025 huggingface dataset:
Stimulation of insulin release by vasopressin in the clonal β-cell line, HIT-T15: the role of protein kinase C
Authors: Hughes S. J. , Carpinelli A. , Niki I. , Nicks J. L. , Ashcroft S. J. H.
Published: December 2008

We have studied the effects of vasopressin and tetradecanoyl phorbol acetate (TPA) on cytosolic free Ca2+ ([Ca2+]i) and insulin release in HIT-T15 β-cells. Saturable binding of [3H] [Arg8]-vasopressin to HIT cell microsomes indicated a single class of receptors with a dissociation constant (Kd) of 2.5 nm and a total number of binding sites (Bmax) equal to 120 fmol/mg protein. [Arg8]-vasopressin (0.1–100 nm) elicited dose-dependent insulin release from HIT cells by up to 25-fold. This increase was dependent on the presence of extracellular glucose and was blocked by omission of extracellular Ca2+ or addition of verapamil. The stimulation was biphasic; a rapid but short-lived large increase in release was followed by a smaller sustained rise. Vasopressin also evoked a marked, concentration-dependent increase in [Ca2+]i which was also biphasic; an initial spike was followed by a sustained elevation. This increase also required glucose and was blocked by the absence of extracellular Ca2+ or the addition of verapamil. Pretreatment of the cells with TPA overnight to deplete protein kinase C activity did not affect the [Ca2+]i or insulin responses to vasopressin. However, short-term exposure to TPA markedly reduced glucose-induced steady-state [Ca2+]i, despite potentiating glucose-stimulated insulin release sevenfold, and blocked the [Ca2+]i increase induced by vasopressin. These inhibitory effects of TPA were absent in protein kinase C-depleted cells and were prevented by staurosporine. TPA had no significant effect on vasopressin-induced insulin release. Vasopressin did not modify the activity of ATP-sensitive K+ channels. These data indicate that vasopressin-induced insulin release is associated with a large increase in [Ca2+]i via increased Ca2+ entry and that this signal is markedly reduced by protein kinase C activation. Thus, protein kinase C does not have a direct role in vasopressin-induced insulin release. Simultaneous addition of vasopressin and TPA promotes insulin release, possibly through increased sensitivity of the secretory system to Ca2+ on activation of protein kinase C by TPA.
Characterization of an ovine glucocorticoid receptor cDNA and developmental changes in its mRNA levels in the fetal sheep hypothalamus, pituitary gland and adrenal
Authors: Yang K. , Hammond G. L. , Challis J. R. G.
Published: December 2008

Fetal sheep tissues possess glucocorticoid receptors (GR), and these change in number during the last two-thirds of gestation. There is, however, no information about developmental changes in tissue GR mRNA levels which might account for alterations in fetal GR content. We have therefore cloned and sequenced a 942 bp GR cDNA from a sheep liver cDNA library, and used it to study the relative abundance of GR mRNA in fetal and neonatal sheep tissues. Analysis of the cDNA revealed a partial sequence of the ovine GR which displayed over 80% identity with residues 143–453 in human GR and 163–472 in rat GR. Furthermore, the first zinc finger motif in these receptors was perfectly conserved among species. The relative abundance of GR mRNA was studied in hypothalami, anterior pituitary glands and adrenals in fetuses at days 60–70, 100–110, 125–130 and at term (approximately 145 days), and in newborn lambs. Total RNA extracts (20 μg) were analysed by Northern blot analysis. A single 5.6kb transcript was detected in all three fetal tissues, and its relative abundance did not change significantly throughout gestation. However, in newborn lambs, levels of GR mRNA increased significantly in the hypothalamus and pituitary gland but decreased to undetectable levels in the adrenal. These tissue-specific changes in the relative abundance of GR mRNA did not correlate with alterations in GR content in fetal tissues, which suggests that the latter may reflect alterations in GR mRNA translation, subsequent modifications and/or GR turnover. In addition, the pattern of developmental changes in GR mRNA content of the adrenal differs from that of the hypothalamus and pituitary gland in neonatal lambs, and indicates that tissue-specific factors may influence GR gene expression in neonatal sheep.
RNA—protein interactions in the 5′ untranslated region of preproinsulin mRNA
Authors: Knight S. W. , Docherty K.
Published: December 2008

A comparison between species of the 5′ untranslated region of preproinsulin mRNA revealed conserved sequences associated with a potential stem—loop structure. The present study was undertaken to determine whether specific protein interactions exist with mRNA sequences involved in the formation or stabilization of this structure in the 5′ untranslated region. 32P-Labelled RNA probes corresponding to sequences from this region were synthesized by an in-vitro transcription reaction and used in electrophoretic mobility shift and u.v.-crosslinking studies with cytoplasmic protein extracts from a number of cell lines. Specific protein—RNA interactions were mapped to a sequence located between nucleotides –21 and –50 upstream of the AUG start codon. A number of proteins of molecular mass 25kDa, 40kDa, 46kDa, 58kDa, 69kDa, 97kDa, 110kDa and 160kDa were specifically crosslinked to this sequence. The observed specific protein—RNA interactions in the 5′ untranslated region may affect the activity of preproinsulin mRNA.
Regulation of steady-state follistatin mRNA levels in rat granulosa cells in vitro
Authors: Michel U. , McMaster J. W. , Findlay J. K.
Published: December 2008

The regulation of steady-state follistatin mRNA levels by different pituitary hormones and peptide factors was examined in granulosa cell cultures derived from diethylstilboestrol-treated immature rats. Cytosolic RNA from cell cultures was prepared by lysis and equal amounts of RNA from all samples were analysed with a solution—hybridization assay using a 32P-labelled antisense probe corresponding to a part of exon 5 together with a part of the 5′ end of exon 6 of the rat follistatin gene. In addition, a specific 35S-labelled probe for cyclophilin was used as an internal standard.

The results show that 5 μg FSH/1 for 24 to 72 h stimulated steady-state follistatin mRNA levels, reaching levels 18·5-fold higher than controls. LH (0·2-100 μg/l) had only minor effects on follistatin mRNA levels in FSH-primed granulosa cells and prolactin, GH and IGF-I did not show any significant effects. Activin raised basal as well as FSH-stimulated steady-state follistatin mRNA levels up to ten- and twofold above controls respectively, whereas epidermal growth factor was found to inhibit FSH-stimulated follistatin mRNA levels in a dose-dependent manner.

It is concluded that follistatin mRNA levels in granulosa cells are regulated by FSH rather than LH, and that the stimulation by FSH can be inhibited by epidermal growth factor but enhanced by activin. Activin alone was also capable of stimulating follistatin mRNA.
Characterization of the extracellular region of the human thyrotrophin receptor expressed as a recombinant protein
Authors: Harfst E. , Johnstone A. P. , Nussey S. S.
Published: December 2008

DNA encoding the N-terminal 415 residues of the human thyrotrophin receptor (predicted to code for the large extracellular region) was introduced into Chinese hamster ovary (CHO) cells using the glutamine synthetase/cytomegalovirus amplifiable expression system, and into E. coli using the pGEX-3X expression vector. Substantial quantities of insoluble fusion protein product resulted from bacterial expression; by Western blot analysis, this was shown to be reactive with anti-receptor antibodies raised against a peptide corresponding to residues 313–330. Immunoreactivity was not retained by the solubilized protein. In eukaryotic expression, several successful CHO transfectants were observed and one (ExG2) was characterized thoroughly. Using agarose-bound Concanavalin A, a glycoprotein with an Mr of approximately 60 000 was detected in a detergent extract of metabolically labelled ExG2 cells, agreeing with the predicted molecular size of 45 000, plus carbohydrate. The same protein could also be detected by immunoprecipitation using the experimental anti-peptide antisera and also sera from patients with Graves' disease. The protein was immunoreactive in Western blot analyses of ExG2 cells using the experimental antisera but not the pathological sera, supporting the view that linear sequences are not sufficient for autoantibody binding. These are the first studies in which visualization of eukaryotically expressed recombinant receptor by such immunological techniques has been possible, presumably because of the higher expression of the glutamine synthetase system.

Surprisingly, the recombinant protein was retained within the cells rather than being secreted. The recombinant protein was very effective at absorbing the adenylate cyclase-stimulating activity of the sera from patients with Graves' disease, but not that of thyrotrophin. This suggests that the large N-terminal extracellular region contains epitopes for stimulatory autoantibodies, but that high affinity thyrotrophin binding requires additional components.
Microheterogeneity of thyroid-stimulating hormone from the pituitaries of euthyroid, hypothyroid and hyperthyroid rats
Authors: Pickles A. J. , Peers N. , Robertson W. R. , Lambert A.
Published: December 2008

The microheterogeneity of pituitary thyroid-stimulating hormone (TSH) is dependent on variations in the hormone's carbohydrate moieties. In this study, changes in the pattern of heterogeneity have been assessed by chromatofocusing, which separates the isospecies on the basis of their isoelectric points (pI). Rats (n = 6 per group) were either untreated or rendered hypo- or hyperthyroid by including in the drinking water either propylthiouracil (0·05% for 8 weeks) or thyroxine (T4; 4 mg/l for 6 weeks) before they were killed at 16 weeks. On autopsy, serum TSH and total T4 were (means±s.e.m.): 2±0·3 μg TSH/l and 64±5 nmol T4/l (control); <1 μg TSH/1 and 133±6 nmol T4/l (hyperthyroid); 58±6 μg TSH/1 and 32±6 nmol T4/l (hypothyroid). The pituitaries were individually homogenized and the TSH isoforms separated by chromatofocusing over a pH range of 7–4. Fractions were assayed for TSH by radioimmunoassay. TSH from the control group was distributed into seven major peaks with pI values of (means ±s.e.m., n=6) 6·9±0·1, 6·6±0·1, 6·2±0·1, 5·8±0·1, 5·5±0·1, 5·2±0·1 and 4·8±0·1; 7±3% of the TSH had a pI of <40. Six peaks of TSH were conserved in the hypothyroid group (with pI values of 6·8±0·1, 6·5±0·1, 6·2±0·1, 5·8±0·1, 5·4±0·1 and 5·2±0·1), and 11±4% of the hormone had a pI of <40. In contrast to the other two groups, only one major peak (with a pI of 5·8±0·1) was detected in the pituitaries from the hyperthyroid group; 13 ± 5% of the TSH had a pI of <40. In the pH range of 5·5– 60, the per cent distribution of TSH was 58±15 (hyperthyroid) compared with 17 ± 3 (hypothyroid) and 22±3 (euthyroid). Above pH 6, only 25±13% of the TSH (hyperthyroid) was present compared with 46±5% (hypothyroid) and 45±5% (euthyroid). Below pH 5·5, the per cent distribution of TSH was 19±5 (hyperthyroid), 37±5 (hypothyroid) and 35±3 (euthyroid). In conclusion, both hyper- and hypothyroidism are associated with changes in the composition of pituitary TSH. This change was most marked in the hyperthyroid group, where there was a selective loss of several isoforms of TSH.
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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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.
The nature and significance of multiple isoforms of the oestrogen receptor in breast tumours
Authors: Puddefoot J R , Baker V A , Bakkers B , Marsigliante S , Barker S , Panahy C , Goode A W , Carpenter R , Vinson G P
Published: December 2008

Oestrogen receptors (ERs) in breast tumours are highly heterogeneous. In previous studies we have shown that at least four isoforms may exist. These migrate in isoelectric focusing (IEF) gels to isoelectric points (pI values) 6·1, 6·3, 6·6 and 6·8. Of these the first (pI 6·1) corresponds to the 8S isoform as detected by sucrose gradient fractionation, while the others all sediment at 4S. In a series of 66 breast tumours it was found that those at pI 6·3 and pI 6·8 were significantly correlated with the presence of progesterone receptors.

To characterize the isoforms more fully, ER isoforms labelled by [3H]oestradiol binding were fractionated by IEF. The results were compared with those obtained after sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) and immunoblotting using the H222 anti-ER monoclonal antibody. In other experiments, tumour ER isoforms were covalently labelled with [ring-3H] tamoxifen aziridine and separated by IEF. The individual isoforms were electroeluted from the IEF gel and further analysed by SDS-PAGE and non-denaturing PAGE. In summary, the evidence shows that the isoforms of pI values 6·3, 6·8 and 6·6 have molecular masses of 50, 65 and 70 kDa respectively. In addition, all three of these isoforms, i.e. the pI 6·3, 6·8 and 6·6 isoforms, could form dimers.

We conclude that the three isoforms sedimenting at 4S have the capacity to form dimers and thus may have the potential for binding to oestrogen response elements in the genome.
Localization of ovine follistatin and α and βA inhibin mRNA in the sheep ovary during the oestrous cycle
Authors: Tisdall D J , Hudson N , Smith P , McNatty K P
Published: December 2008

The sites of follistatin and α and βA inhibin gene expression were examined by in situ hybridization in sheep ovaries during the early and mid-luteal phases (days 3 and 10) of the oestrous cycle and a prostaglandin F2α (PGF2α)-induced follicular phase.

Follistatin mRNA was detected in the granulosa cells of preantral, antral and early atretic follicles at all stages of the oestrous cycle, and in the corpora lutea at the early and mid-luteal stages of the cycle. However, only low levels of expression of follistatin were observed in the presumptive preovulatory follicle at 56 h after treatment with PGF2α.

Both α and βA inhibin were shown to be expressed in ovaries at all stages of the oestrous cycle. In situ hybridization localized α subunit mRNA to the granulosa cells of most, but not all, healthy antral follicles, and to no other ovarian cell type. In contrast, expression of the βA subunit was confined to a few medium-to-large healthy antral follicles. In antral follicles expressing βA inhibin, mRNAs for α inhibin and follistatin were always detected, but the converse was not true. Unlike follistatin, no α and βA inhibin expression was seen in preantral follicles, developing corpora lutea, or follicles undergoing atresia.

These results show that, in the adult sheep ovary, follistatin gene expression is a constitutive event in all growing follicles from the early preantral stage, and also provide indirect evidence of the involvement of follistatin, but not inhibin or activin, in the early stages of ovarian follicle development in sheep.
Translocation of protein kinase C isozymes in lactotroph-enriched rat anterior pituitary cell cultures: differential effects of substance P and phorbol ester
Authors: Mau S E , Særmark T , Vilhardt H
Published: December 2008

Translocation of protein kinase C (PKC) from the cytosol to the plasma membranes is believed to reflect activation of the enzyme. We have studied translocation of PKC in lactotroph-enriched anterior pituitary cell cultures by measuring the incorporation of γ-32P from [γ-32P]ATP into a synthetic peptide substrate, MBP4–14, and by immunoblotting of PKC isozymes. Using cells permeabilized with digitonin the effects of PKC cofactors on the distribution of the enzyme were studied. Ca+ (50 nm) and dioctanoyl-sn-glycerol had no effect when tested alone, but in combination they caused a redistribution of PKC from the soluble to the particulate fraction. Arachidonic acid needed Ca+ to induce translocation of PKC, while being ineffective under Ca2+-free conditions. Western blot analysis of partly purified PKC from lactotroph-enriched pituitary cells revealed the presence of the α, β, δ, and ζ isozymes. 12-O-Tetradecanoylphorbol 13-acetate (TPA) and substance P displayed different patterns of redistribution of PKC isozyme immunoreactivity from soluble to membrane-attached forms. Thus, TPA induced time- and dose-dependent (mean effective concentration (EC50)=1 nm) translocation of the α, β and δ species, while substance P stimulated time- and dose-dependent (EC50=1 nm) redistribution of the α and β isozymes. ζ subtype immunoreactivity could not be translocated by either agonist; neither could the immunoreactivity of ζ be down-regulated by long-term treatment (24 h) with TPA.

The results indicate that simultaneous activation of phospholipases C and A2 induces a synergistic activation of PKC. Finally it is suggested that substance P may exert some of its effects in lactotrophs by translocation of PKC isozymes α and β.
Effect of in vivo oestradiol treatment on cell-free transcription in trout liver nuclear extracts
Authors: Delaunay F , Pakdel F , Valotaire Y
Published: December 2008

In order to perform later studies on the transcriptional regulation of hormone-dependent genes in fish liver, we firstly examined the potential of trout liver nuclear extracts in a cell-free transcription system. As reporter genes, we used DNA sequences without G (G-free cassettes) under the control of three promoters derived from the 5′ flanking sequence of the Xenopus vitellogenin B1 gene; two of them were responsive to the oestrogen receptor (ER) through oestrogen responsive elements (ERE). Maximal transcriptional activity was obtained within a range of 40–130 μg protein per extract depending on the extract preparation. Transcription was maximal in reactions carried out at 25 °C.

Similar transcriptional activities for the three promoters were observed when transcription was performed in extracts from untreated male trout. In contrast, we observed a 4·5- to 6-fold increase in the transcription with ERE-containing promoters in comparison with that with the minimal promoter bearing only a TATA box when extracts from oestradiol-treated male trout were used. This effect was correlated with the increase in the nuclear ER concentration induced by in vivo hormonal treatment. This enhanced transcription was specifically inhibited by the addition of a 25- to 100-fold excess of ERE oligonucleotide competitor.

These data demonstrated, therefore, that transcription was ERE-dependent in this system and suggest strongly that it was mediated by the trout ER. Addition of oestradiol or the anti-oestrogens hydroxytamoxifen or ICI 164384 had no effect on the transcriptional activity of the two ERE-containing promoters, indicating that transcription was hormone-independent in trout liver nuclear extracts.
Dual activity of human pituitary thyrotrophin isoforms on thyroid cell growth
Authors: Medri G , Sergi I , Papandréou M-J , Beck-Peccoz P , Verrier B , Ronin C
Published: December 2008

Alkaline (pI 8·6–7·5) and neutral (pI 7·0–6·0) isoforms of human TSH have been isolated from a highly purified intrapituitary preparation by isoelectric focusing and compared for their respective actions on thyroid cell proliferation. Both TSH isoforms displayed the same ability to bind to porcine thyroid membranes as the original hormone preparation, indicating a similar recognition at the receptor sites. Alkaline forms showed a higher potency in inducing either cyclic AMP (cAMP) production or [3H]thymidine incorporation in FRTL-5 cells (half-maximal effective doses (ED50 values)=0·25 and 0·29 nm respectively) compared with their neutral counterparts (ED50 values=0·66 and 0·70 nm respectively). Increasing the concentration of alkaline forms in the presence of a half-maximal concentration of neutral TSH resulted in a profound inhibition of cell growth without a significant change in cAMP. Conversely, increasing the amount of neutral forms in the presence of a half-maximal dose of alkaline TSH resulted in an additive response for cAMP production but not in cell proliferation.

To assess whether glycosylation might be responsible for the variation in hormone action, both alkaline and neutral TSH isoforms were tested for recognition of their carbohydrate chains by concanavalin A (Con A) and ricin. No major difference was found in binding to Con A, indicating that the contribution of carbohydrates to changes in hormone pI was not related to core branching. Very few galactose residues were accessible in either hormone fraction since little binding to ricin was observed. Isoelectric focusing of TSH forms before and after neuraminidase treatment revealed that neutral forms had a higher sialic acid content than alkaline TSH.

In conclusion, the current findings show that TSH isoforms differentially affect cAMP production and cell growth. TSH fractions with a high sialic acid content and a low mitogenic activity behave as antagonists to the more active forms for cell proliferation. It is suggested that physiological control of TSH action at the thyroid gland may reside in the respective amounts of various TSH forms which, once bound to their receptor, can induce variable activation of post-receptor events while controlling cell proliferation.
Isolation and characterization of ovine IGFBP-4: protein purification and cDNA sequence
Authors: Carr J M , Grant P A , Francis G L , Owens J A , Wallace J C , Walton P E
Published: December 2008

Three different molecular mass forms of IGF-binding proteins (IGFBPs) were purified from ovine plasma by IGF-I affinity chromatography and reverse-phase HPLC: a 46 kDa doublet and 29 kDa and 24 kDa forms. Amino-terminal sequence analysis confirmed that these proteins were ovine (o)IGFBP-3 (46 kDa) and two molecular size variants of oIGFBP-4. oIGFBP-3 and the 29 kDa form of oIGFBP-4 were shown to be N-glycosylated. Isoelectric points were determined to be at approximately pH 6 for oIGFBP-3 and at pH 7 and pH 7·5 for the 29 and 24 kDa forms of oIGFBP-4 respectively. The two different molecular mass variants of oIGFBP-4 had similar IGF-binding properties. Compared with human IGFBP-3 and oIGFBP-3, the two variants of oIGFBP-4 exhibited lower relative binding to amino-terminally modified IGF-I analogues in a competitive IGF-binding assay. The full protein sequence of oIGFBP-4, as deduced from the cDNA sequence, showed a high degree of identity with rat (90%), human (96%) and bovine (98%) IGFBP-4. The cDNA sequence also showed homology over regions of the 3′ non-coding sequence, particularly in comparison with bovine IGFBP-4 (96%). Northern analysis of mRNA for oIGFBP-4 indicated a 26 kb major transcript and two minor transcripts of approximately 21 and 1·8 kb. oIGFBP-4 mRNA transcripts were detected in adult ewe liver>kidney>lung>>heart and also in several fetal tissues, thus suggesting tissue-specific and developmental regulation. The availability of purified oIGFBP-4 and oIGFBP-3 as well as DNA probes for oIGFBP-4 will enable further study of the properties and functions of these proteins, as well as the establishment of specific assays for these IGFBPs.
Production and characterization of recombinant chicken insulin-like growth factor-II from Escherichia coli
Authors: Upton Z , Francis G L , Kita K , Wallace J C , Ballard F J
Published: December 2008

Recombinant chicken (c)IGF-II has been produced in Escherichia coli after first modifying a plasmid that coded for a human (h)IGF-II fusion protein. The cIGF-II fusion protein, deposited in bacterial inclusion bodies, was dissolved under reducing conditions, desalted, subjected to anion-exchange chromatography and refolded. Recombinant cIGF-II was then released from the fusion protein using a genetically engineered serine protease and purified to homogeneity by reverse-phase HPLC. In vitro analysis of recombinant cIGF-II revealed differences between cIGF-II and its human counterpart. Recombinant cIGF-II was less potent than hIGF-II in stimulating protein synthesis in rat myoblasts. This appeared to be due to a decreased affinity for the type-1 IGF receptor. The human and chicken peptides were similar, however, in studies assessing binding to the type-2 IGF receptor and to IGF-binding proteins. Moreover, recombinant cIGF-II and hIGF-II were equipotent in both biological and receptor binding studies in chick embryo fibroblasts, suggesting that there may be a difference between mammalian and avian type-1 IGF receptors.