bluuebunny/crossref_metadata_2025 huggingface dataset:
Nuclear proteins from the rat pituitary gland bind to regulatory sequences of the thyrotrophin-β gene
Authors: Ahlquist J. A. O. , Franklyn J. A. , Ramsden D. B. , Sheppard M. C.
Published: December 2008

Interactions between DNA sequences and nuclear proteins are important in the regulation of gene expression. We have applied a gel retardation method to examine the binding of nuclear proteins from the rat pituitary gland to regulatory sequences of the rat TSH β-subunit gene. Binding between nuclear protein and DNA is demonstrated by an alteration in electrophoretic mobility of the DNA. A 0·4 m NaCl fraction of pituitary nuclear proteins bound to a fragment of DNA containing the promoter region of the TSH-β gene, but not to plasmid DNA or insulin cDNA of comparable size. This nuclear protein fraction also bound to fragments of DNA containing sequences from the 5′-flanking region of the gene; binding of nuclear proteins was evident as far as 1 kb 5′ to the structural gene. Each of these regions of the TSH-β gene formed a number of distinct complexes with the nuclear protein extract, distinguished by their differing binding affinities in the presence of poly(dI-dC), and by their electrophoretic mobility. These findings suggest that transcriptional regulation of the rat TSH-β gene may be mediated by interaction of a number of nuclear proteins with regulatory sequences of DNA up to 1 kb upstream of the transcriptional start site of the gene.
Effects of benextramine on the adrenergic inhibition of insulin secretion in isolated rat pancreatic islets
Authors: Hurst R. D. , Chan S. L. F. , Morgan N. G.
Published: December 2008

Insulin secretion from isolated rat islets of Langerhans in the presence of 4 mm glucose averaged 2·26 ± 0·20 (s.e.m.) ng/islet per 90 min and was significantly (P<0·001; n=30) increased to 3·28 ± 0·21 ng/islet per 90 min by the covalent α-adrenoceptor antagonist benextramine (10 μm). Glucose (20 mm) also increased the secretion rate (to 6·24 ± 6·0 ng/islet per 90 min) but, under these conditions, the response was not further enhanced by benextramine. Clonidine and noradrenaline (1 nm–10 μm) each caused dose-dependent inhibition of glucose-induced insulin secretion which was maximal at 1 μm. Benextramine, when added simultaneously with the agonist, relieved, in a dosedependent manner, the inhibition of secretion induced by either clonidine or noradrenaline with similar sensitivity. Even after a 30-min preincubation with benextramine the antagonist failed to differentiate between noradrenaline, adrenaline and clonidine with respect to inhibition of insulin secretion. In contrast to its effects on adrenergic responses, short-term treatment with benextramine did not significantly affect muscarinic—cholinergic receptor-mediated 45Ca2+ efflux from rat islets of Langerhans perifused in Ca2+-depleted medium. These data suggest that benextramine does not differentiate between clonidine and noradrenaline in rat islets of Langerhans but that it does show preference for α-adrenoceptors in this tissue.
In-vitro biosynthesis of multiple preproglucagons results from acetylation of the primary translation products
Authors: Okun M. M. , Warren T. G. , Shields D.
Published: December 2008

In-vitro translation of anglerfish islet mRNA revealed three glucagon precursors (preproglucagons): one with Mr 16 000 and two with Mr 14 000. The two Mr 14 000 precursors were well separated upon isoelectric focusing gels (pI values of 7·2 and 7·3), but had identical peptide maps. Translation of hybrid-selected Mr 14 000 preproglucagon mRNA in the presence of microsomal vesicles revealed that both precursors were processed to the same proglucagon. Northern blot analysis detected two mRNA species encoding Mr 14 000 precursor. A full-length Mr 14 000 preproglucagon cDNA was subcloned into a transcription vector, and coupled in-vitro transcription—translation was performed; surprisingly, both Mr 14 000 precursors were synthesized. To test whether acetylation of the free amino terminus generated the more acidic precursor, acetylase activity was partially inactivated with the inhibitor S-acetonyl-CoA, and acetyl-CoA was depleted by addition of oxaloacetate and citrate synthetase. Under these conditions, the level of the most basic preproglucagon was greatly enhanced, but when exogenous acetyl-CoA was added, the acidic form predominated. We conclude that acetylation generates the acidic precursor, and we discuss the implications of our findings for the biogenesis of other peptide hormones.
Immune induction of major histocompatibility complex antigens on a human thyroid cell line (SGHTL-34)
Authors: King S. L. , Whitley G. St J. , Page S. , Nussey S. S. , Johnstone A. P.
Published: December 2008

Expression of major histocompatibility complex antigens by epithelial cells may play a role in the aetiology of autoimmune disorders. We have studied the effect of γ-interferon on SGHTL-34, a human thyroid cell line which constitutively expresses class I but not class II antigens. γ-Interferon induced the expression of class II and increased the expression of class I molecules (assessed by flow cytofluorimetry) in a dose-dependent manner. Thyrotrophin or phytohaemagglutinin had no effect on either class I or class II expression. However, a supernatant from phytohaemagglutinin-stimulated peripheral blood mononuclear cells, containing 6400 U γ-interferon/ml, was an effective inducer of both class I and class II antigens. These data clarify earlier studies using primary thyroid cultures, which are contaminated with cells of the immune system.
Thyrotrophin, forskolin and ionomycin increase cathepsin B mRNA concentrations in rat thyroid cells in culture
Authors: Phillips I. D. , Black E. G. , Sheppard M. C. , Docherty K.
Published: December 2008

In order to study the regulation of cathepsin B expression in the thyroid, cathepsin B mRNA concentrations were measured in rat thyroid cells (FRTL5) in culture. Northern blot analysis demonstrated that cathepsin B mRNA concentrations were increased in FRTL5 cells cultured for up to 6 days in TSH. The effect of TSH on cathepsin B mRNA concentrations was dose dependent over the range 25–150 μunits/ml. Cytoplasmic dot-blot analysis was used to characterize this effect further. The TSH-induced increase in cathepsin B mRNA concentrations (approximately fivefold over that in untreated cells) was partially mimicked by forskolin (approximately threefold) and ionomycin, while phorbol ester decreased cathepsin B mRNA concentrations. Similar changes were observed for thyroglobulin and actin mRNA concentrations. TSH had no effect on cathepsin B enzymatic activity or immunoreactive protein concentration. These results demonstrate (1) that cathepsin B expression in the thyroid is regulated in parallel with that of thyroglobulin and actin, and (2) that cyclic AMP- and Ca2+-dependent processes stimulate gene expression, while phorbol ester treatment inhibits gene expression in FRTL5 cells.
Effects of antioestrogens on the proliferation of MCF-7 human breast cancer cells
Authors: Wakeling A. E. , Newboult E. , Peters S. W.
Published: December 2008

Non-steroidal antioestrogens, such as tamoxifen, inhibit the growth of human breast cancer cells. The experiments described here compare and contrast the efficacy of tamoxifen and the 'pure' antioestrogen, ICI 164384, on the inhibition of proliferation of MCF-7 cells.

Previous studies have shown that ICI 164384 has a greater maximal inhibitory effect than conventional antioestrogens on the growth of MCF-7 cells. Both types of compound block progression of cells through the cell cycle in the early G1 phase. These studies have been extended to measure the population distribution of antioestrogen-treated cells by the use of two-parameter flow cytometry. ICI 164384 proved to be more effective than tamoxifen in decreasing the proportion of actively growing cells in an asynchronous population.

In cells grown in the complete absence of exogenous oestrogens, growth was stimulated by oestradiol, insulin, insulin-like growth factor-I (IGF-I) or transforming growth factor-α (TGF-α). The potent metabolite of tamoxifen, trans 4′-hydroxytamoxifen (4′-OHT), alone also stimulated growth, whereas ICI 164384 did not. Oestradiol and insulin added together demonstrated a clear synergistic enhancement of cell growth. Correspondingly, the stimulatory effect of 4′-OHT on growth was magnified in the presence of insulin, and a combination of ICI 164384 with insulin revealed a much weaker stimulatory action of the 'pure' antagonist. For both compounds the interaction with insulin was complex and characterized by a bell-shaped dose—response curve. However, for 4′-OHT at all concentrations in the range 1 pm–1 μm in the presence of insulin, cell numbers were greater than in cultures exposed to insulin alone. This was not the case for ICI 164384 which suggested that differences in efficacy may be due to interactions between oestrogen and growth factor-mediated mechanisms. Furthermore, ICI 164384 was more effective in inhibiting the action of IGF-I and TGF-α alone or in combination, although both antioestrogens produced a partial blockade of growth factor responses in the complete absence of oestradiol.

It is concluded that the difference in efficacy between partial agonist and 'pure' antagonist antioestrogens to inhibit growth in vitro is consistent with the difference in the pharmacological profile of these compounds. The absence of stimulatory activity of ICI 164384 is of particular significance in reducing to a minimum the synergistic interaction between oestrogens and insulin. In addition, the partial blockade of the effect of IGF-I and/or TGF-α by antioestrogens in the complete absence of oestradiol, where ICI 164384 is also more effective than 4′-OHT, suggests that novel mechanisms of growth inhibition may be involved.
Interleukin-1 induces a pertussis toxin-sensitive increase in diacylglycerol accumulation in mouse thymoma cells.
Authors: Dobson P.R.M. , Plested C.P. , Jones D.R. , Barks T. , Brown B.L.
Published: December 2008

The mechanism of action of the cytokine, interleukin-l (IL-1), has been investigated. Mouse thymoma (EL4 6.1) cells were preincubated with [3H]-glycerol and then incubated with recombinant IL-1β for varying periods. Interleukin-1 caused a rapid increase in diacylglycerol production (approx. 2 fold at 30 sees). This reproducible enhancement of diacylglycerol accumulation was abolished by pretreatment of the cells with pertussis toxin. Interestingly, a similar IL-1 induced increase in diacylglycerol was observed when the cells were preincubated with [3H]-myristic acid. These results appear to suggest a novel mode of action of interleukin-1 which involves a G-protein mediated breakdown of a membrane lipid resulting in the production of diacylglycerol. It is suggested that one possible candidate for this parent lipid may be a phosphatidylinositol glycan.
Early regulation of c-myc mRNA by 1,25-dihydroxyvitamin D3 in human myelomonocytic U937 cells
Authors: Karmali R. , Bhalla A. K. , Farrow S. M. , Williams M. M. , Lal S. , Lydyard P. M. , O'Riordan J. L. H.
Published: December 2008

The effects of 1,25-dihydroxyvitamin D3 (1,25-(OH)2D3; 10 nmol/1) on the human monomyelocytic cell line U937 were investigated. Addition of 1,25-(OH)2D3 led to a decrease in cell proliferation which fell at 72 h to 67·8 ± 4·3 % (mean±s.e.m.) of control values. The presence of CD14, a surface marker found on mature monocytes/macrophages but not on U937 cells, was detectable as early as 18 h and peaked at 48 h, when 63·6 ± 4·2% of the cells were positive. However, changes in c-myc mRNA levels were detected earlier, starting within 4 h of exposure to the hormone and being reduced to 38±8·2% of control values of 24 h. These effects were reversible after removal of the hormone, with the same sequence of events seen following addition of the hormone. There was first an increase in c-myc mRNA levels, starting within 2 h and reaching control values by 24 h. These changes were followed by loss of CD14 which became undetectable after 72 h. Proliferation recovered slowly and incompletely, since it was 81·7 ± 0·7% of control after 72 h. A constant reciprocal relationship between c-myc mRNA and CD14 levels was found both in the presence and after removal of 1,25-(OH)2D3.

Regulation of U937 cell proliferation and maturation by 1,25-(OH)2D3 is thus preceded by early modulation of c-myc mRNA.
Decreased expression of hepatic epidermal growth factor receptor gene in diabetic mice
Authors: Kasayama S. , Yoshimura M. , Oka T.
Published: December 2008

The expression of hepatic epidermal growth factor (EGF) receptor gene was studied in genetically diabetic (C57BL/KsJ db/db) mice and streptozotocininduced diabetic mice. The binding of 125I-labelled EGF to hepatic membrane preparations of genetically diabetic mice was only 35% of that of non-diabetic mice. Levels of EGF receptor messenger RNAs (10 and 6 kb) in the liver of the diabetic animals were also reduced by about 75%. In streptozotocin-induced diabetic mice, levels of hepatic EGF binding and messenger RNAs for EGF receptor were decreased to 27 and 30% of control levels respectively, at 5 weeks after injection of the drug. There were, however, no significant differences in levels of messenger RNAs for the structural protein β-actin in the liver. In addition, levels of EGF receptor messenger RNAs in the kidney were similar between control and the two kinds of diabetic mice. Daily administration of insulin to the streptozotocin-induced diabetic mice increased the hepatic levels of EGF receptor messenger RNAs to almost normal levels. These results indicate that EGF binding to its receptor decreases in the liver of diabetic mice, involving alterations in the level of EGF receptor messenger RNAs, and that insulin is important for the regulation of EGF receptor gene expression in the liver but not in the kidney.
Pro-opiomelanocortin gene expression and peptide secretion in human small-cell lung cancer cell lines
Authors: White A. , Stewart M. F. , Farrell W. E. , Crosby S. R. , Lavender P. M. , Twentyman P. R. , Rees L. H. , Clark A. J. L.
Published: December 2008

Expression of the RNA coding for the ACTH—β-lipotrophin precursor, pro-opiomelanocortin (POMC), has been demonstrated in five human small-cell lung cancer (SCLC) cell lines. Using Northern and slot-blot hybridization analysis of RNA and a bovine POMC cDNA as probe, the processed POMC RNA from SCLC cells was found to be approximately 1350 nucleotides in length, which is larger than that found in the normal human pituitary. Expression of the POMC gene was confirmed by measurement of ACTH precursors secreted by the cells, using a novel two-site immunoradiometric assay based on monoclonal antibodies, which directly quantifies both POMC and pro-ACTH but does not recognize ACTH. Levels of POMC in medium accumulated throughout the growth of the cells, in contrast to POMC RNA which showed a relatively constant level of expression. We conclude that human SCLC cell lines are valuable models for studying the aberrant expression and regulation of the human POMC gene.
The androgen-dependent rat prostatic binding protein: comparison of the sequences in the 5′ part and upstream region of the Cl and C2 genes and analysis of their transcripts
Authors: Claessens F. , Dirckx L. , Delaey B. , Decourt J.-L. , Hemschoote K. , Peeters B. , Rombauts W.
Published: December 2008

The complete gene encoding the polypeptide Cl of the complex androgen-controlled prostatic binding protein was isolated from a rat genomic library. A new genomic fragment (C2B) containing only the 5′ part of a C2-related gene was also purified. The segments containing exon 1 and a large part of the adjacent sequences were analysed and compared with the corresponding region of the C2A gene which has been completely sequenced previously. The high structural similarity extending over a large part of all three genomic fragments suggests the duplication of a common ancestral gene, followed by a more recent duplication of the C2-coding region. However, since the structural similarity upstream of position − 150 between C2A and C2B abruptly disappears and no transcripts specific for the C2B region can be detected in prostate RNA, we propose that at a later stage in evolution the C2B region was disrupted and inactivated. Despite the common origin and the similar regulation of the two active genes, Cl and C2A, the only obvious conserved structural element is the homopurine stretch located at position −400, although sequence motifs resembling steroid hormone response elements are present at several locations.
Expression and partial purification of human pro-opiomelanocortin in Escherichia coli
Authors: Grewal T. S. , Lowry P. J. , Savva D.
Published: December 2008

A large portion of the human pro-opiomelanocortin (POMC) peptide corresponding to amino acid residues 59–241 has been cloned and expressed in Escherichia coli. A 1·0 kb DNA fragment encoding this peptide was cloned into the expression vectors pUC8 and pUR291. Plasmid pJMBG51 (a pUC8 recombinant) was found to direct the expression of a 24 kDa peptide. The recombinant pUR291 (pJMBG52) was shown to produce a β-galactosidase fusion protein of 140 kDa. Western blot analysis showed that both the 24 kDa and 140 kDa peptides are recognized by antibodies raised against POMC-derived peptides. The β-galactosidase fusion protein has been partially purified from crude E. coli cell lysates using affinity chromatography on p-aminobenzyl-1-thio-β-d-galactopyranoside agarose.
Growth hormone regulates the rodent hepatic epidermal growth factor receptor at a pretranslational level
Authors: Johansson S. , Husman B. , Norstedt G. , Andersson G.
Published: December 2008

Recent studies have implicated the involvement of pituitary factor(s) in the regulation of hepatic epidermal growth factor receptor (EGF-R) levels. In the present study the possible role of GH as a regulator of EGF-R has been examined by measuring hepatic EGF-R mRNA and EGF binding in intact and GH-deficient rats and mice before and after administration of GH. Using a human EGF-R probe, 10·5 and 6·0 kb transcripts were detected in mouse and rat liver by Northern gel analysis. EGF-R mRNA was quantified by solution hybridization, and EGF binding determined by incubation of 125I-labelled EGF with a low-density membrane fraction.

Levels of hepatic EGF-R mRNA and binding of EGF in female rats were about two-thirds of those in male rats. GH-deficient (lit/lit) male and female mice had approximately 10 and 25% respectively of the levels of EGF-R mRNA and EGF binding of intact male and female mice. Furthermore, hypophysectomized rats exhibited a reduced level of EGF-R mRNA and EGF binding, corresponding to about 20% of the levels detected in intact male rats. When hypophysectomized male and female rats received recombinant human GH (hGH) either as intermittent injections or by continuous infusion using osmotic minipumps, the EGF-R mRNA and EGF binding levels increased to about half those of the intact male animals. No differences between intermittent or continuous administration of hGH on the induction of EGF-R mRNA or EGF binding could be seen. The correlation between mRNA and binding levels suggests regulation at a pretranslational level.

There was a reduction of EGF-R mRNA to female levels when intact male rats were given hGH. An additional reduction of EGF binding levels was seen in both intact male and female rats exposed continuously to GH. This may indicate additional translational and/or post-translational regulatory mechanisms.
Dual effects of thyrotrophin-releasing hormone (TRH) on K+ conductance in frog pituitary melanotrophs. TRH-induced α-melanocyte-stimulating hormone release is not mediated through voltage-sensitive K+ channels
Authors: Louiset E. , Cazin L. , Lamacz M. , Tonon M.-C. , Vaudry H.
Published: December 2008

Modulation of the activity of K+ channels by TRH and the possible involvement of this modulation in TRH-induced release of α-MSH were studied in cultured frog melanotrophs, using patch-clamp and perifusion techniques. Pars intermedia cells were enzymatically dispersed and cultured in Leibovitz medium. In order to test the viability of cultured cells, the amount of α-MSH released into the medium was measured by radioimmunoassay every day for 1 week of culture. The total amount of α-MSH released during the first 4 days of culture was 8·6 times higher than the intracellular content of α-MSH on day 1. Melanotrophs were identified by an indirect immunofluorescence technique using a specific antiserum to α-MSH. Recordings obtained in whole-cell, cell-attached and excised patch-clamp configurations showed that TRH induced a transient polarization concomitant with an increase in the probability of opening of Ca2+-activated K+ channels. This transient response was followed by a depolarization accompanied by an enhanced frequency of action potential discharge. TRH also induced a decrease in voltage-dependent K+ conductance. Application of tetraethylammonium, a K+ channel blocker, depolarized the cells and increased the basal secretory level without noticeable changes in TRH-evoked α-MSH release.

These results demonstrate that the neuropeptide TRH both stimulates Ca2+-sensitive K+ channels and inhibits voltage-dependent K+ current in pituitary melanotrophs. Our data indicate that TRH-induced secretion of α-MSH is not a direct consequence of the lowering of K+ conductance. It thus appears that basal and TRH-induced α-MSH release occur through distinct pathways; the spontaneous release of α-MSH is probably linked to membrane potential, while modulation of the electrical activity is not directly involved in TRH-induced activation of the secretory process.
Structural characteristics of growth hormone receptors on mouse 3T3 cells having different biological responses to growth hormone
Authors: Uchida E. , Hayakawa T. , Niimi S. , Tanaka A. , Morikawa M.
Published: December 2008

Cultured 3T3-F442A preadipocytes are able to undergo GH-promoted differentiation into adipocytes. The relationship between the structure and function of GH receptors on 3T3 cells (3T3-F442A preadipocytes, differentiated adipocytes and 3T3-C2 cells, which vary in susceptibility to adipose conversion or with respect to carbohydrate and lipid metabolism) was studied by the covalent cross-linking of 125I-labelled human (h) GH to intact cells with the bifunctional reagent disuccinimidyl suberate. When preadipocytes were cross-linked and analysed using sodium dodecylsulphate-polyacrylamide gel electrophoresis, a prominent 125I-labelled hGH-receptor complex of Mr 130 000 was observed along with minor complexes (Mr 300 000, 230 000 and 60 000) on autoradiography. Non-reducing—reducing two-dimensional gel electrophoresis revealed that the higher molecular weight complexes also contained the Mr 130 000 complex. Neuraminidase and tunicamycin treatment demonstrated that the GH receptor on F442A preadipocytes is a sialo-glycoprotein with N-linked carbohydrate chains. When the differentiated 3T3-F442A adipocytes and 3T3-C2 cells (a sub-line with no susceptibility to adipose conversion with GH) were examined in the same way as 3T3-F442A preadipocytes, no differences were observed in the specificity of GH binding and in the molecular size of the 125I-labelled hGH-receptor complexes and their glycosylation characteristics. This suggests that the structural characteristics of the GH receptor are closely related in each cell type, but that the hormonal signals produced after GH binding to the receptor may cause different effects according to the cell type.
Inducible anti-sense RNA for angiotensinogen in stably transformed hepatoma cell lines
Authors: Clouston W. M. , Lloyd C. J. , Richards R. I.
Published: December 2008

Angiotensinogen mRNA is found in many extrahepatic tissues, where it may participate in local angiotensin-generating systems. In this study we explore the feasibility of using anti-sense RNA to decrease angiotensinogen production in rat H4IIEC3 hepatoma cells. An amplifiable shuttle vector was modified to allow the production of high levels of stable anti-sense RNA from two regions of the mouse angiotensinogen gene under the control of the inducible sheep metallothionein promoter. Stably transformed, clonal cell lines expressing anti-sense RNA for angiotensinogen were isolated after selection with the aminoglycoside G418. Subsequently, the number of chromosomally integrated copies of the angiotensinogen anti-sense constructs was coamplified by methotrexate selection for dihydrofolate reductase activity carried on the shuttle vector. With a 20- to 30-fold induction of the anti-sense RNAs, the target angiotensinogen mRNA level was reduced to 25–30% of control values. The specificity of this effect was confirmed by showing no decrease in either β-tubulin or neomycin phosphotransferase mRNA levels. Using tissue-specific promoters, it should be possible to direct these effects to specific organs in transgenic mice. However, in agreement with results from other groups, our findings suggest that it will not be possible to eradicate completely the target gene product using the anti-sense RNA strategy.
Molecular cloning of cDNA for porcine prolactin precursor
Authors: Kato Y. , Hirai T. , Kato T.
Published: December 2008

Porcine prolactin cDNA clones were screened using antiserum against ovine prolactin from a cDNA library of porcine anterior pituitary, and their nucleotide sequences were determined by the chain-termination method. The nucleotide sequence of the 5′ untranslated region and part of the signal peptide region were determined by direct RNA sequencing with reverse transcriptase. The composite sequence of 957 nucleotides showed a signal sequence of 30 amino acids and a further 199 amino acids corresponding to the mature prolactin molecule. The predicted sequence confirmed the amino acid sequence determined previously by direct protein analysis, except for one amide form at residue 122 (Gln instead of the reported Glu). Northern blot analysis showed that the length of the porcine prolactin mRNA was about 1·1 kb. The porcine prolactin amino acid sequence showed 81, 80, 64, 62, 80 and 31% homology with human, bovine, rat, mouse, chick and salmon forms respectively. The identical amino acid residues showed marked clustering in four domains, two of which are highly conserved throughout a wide range of species. The hydropathy and secondary structure of porcine prolactin were analysed and compared with those of porcine GH, which shares the same ancestral gene. The two highly conserved regions of both hormones showed similar hydrophilicity, and the predicted secondary structures indicated that these regions in each hormone form different structures with differences in extension of the hydrophilic residues outside the molecule.
Partial cDNA sequence for the donkey chorionic gonadotrophin-β subunit suggests evolution from an ancestral LH-β gene
Authors: Leigh S. E. A. , Stewart F.
Published: December 2008

A 246 bp cDNA clone representing the C-terminal region of the donkey (Equus asinus) chorionic gonadotrophin (CG)-β subunit was isolated from a placental library. The transcript contained the 3′ untranslated region and 42% of the CG-β subunit coding region (amino acid residues 85–146 of the mature peptide). Comparison of the deduced donkey amino acid sequence with the published horse CG-β subunit protein sequence (where they overlapped) revealed an overall homology of 61%. However, most of the differences were in the C-terminal extension, which is thought not to be important for gonadotrophic activity, and appeared to be due to two nucleotide insertions in the donkey sequence (compared with a deduced horse nucleotide sequence) leading to a reading-frame shift. Amino acid homology in the disulphide 'core' region was 81%. Some of the differences in this region were in the 'determinant loop' (residues 93–100) and these are interpreted in relation to the observed biological activities of horse and donkey CG.

The deduced amino acid sequence of the donkey cDNA indicated that it was larger than the majority of gonadotrophin-β subunits due to a C-terminal extension. Primate and horse CG (and horse LH) β subunits have analogous C-terminal extensions. The extension in the donkey subunit is 25 amino acid residues in length, compared with 28 in the horse and 24 in man. Comparisons with other available subunit DNA sequences indicated that, like the human CG-β gene, the donkey gene probably evolved from an ancestral LH-like β gene, following nucleotide deletions that allowed readthrough into previously untranslated DNA. Furthermore, both the human and donkey CG-β genes make use of the original LH polyadenylation sequence AAUAAA for translational termination and polyadenylation. We conclude that the C-terminal extension arose independently in equids and primates but through similar mechanisms.
Poly(A) tail length of oxytocin- and lysine vasopressin-encoding mRNAs increases during development in the porcine hypothalamus
Authors: Rehbein M. , Richter D.
Published: December 2008

Lysine vasopressin- and oxytocin-encoding mRNAs have been analysed in the developing hypothalamus of the pig. The two hormone-encoding mRNAs were first detectable on fetal day 49 by Northern blot analysis. Whereas RNase mapping revealed identical transcripts throughout the developmental stages studied, Northern blots showed that the early transcripts appeared to be shorter (by 100–200 nucleotides) and more heterogeneous in size than those of later stages. This developmentally related length polymorphism was shown to be due to different poly(A) lengths and was abolished by removal of the poly(A) tails with RNase H. These results indicate that maturation of neurones in the developing porcine hypothalamus is accompanied by an increase in length of the poly(A) tail of vasopressin and oxytocin mRNAs.
Molecular cloning, sequence analysis and expression of putative chicken insulin-like growth factor-I cDNAs
Authors: Fawcett D. H. , Bulfield G.
Published: December 2008

A cDNA library was constructed from mRNA isolated from the liver of a 5-week-old female broiler chicken; at this age the level of insulin-like growth factor-I (IGF-I) mRNA was expected to be high. Three clones, of sizes 23, 0·86 and 0·2 kb, were isolated by using a homologous human (IGF-I) probe. The DNA sequence of these clones has been determined and the potential amino acid sequence deduced. The sequence of the mature chicken IGF-I peptide shows a high degree of homology with IGF-I from other species, providing evidence for the identity of these clones. Alternative splicing of the chicken IGF-I mRNA has been found in the region potentially encoding the leader peptide. This may give rise to two forms of prepeptide, differing in the length and nature of their leader peptide. The 086 kb cDNA has been used as a probe to Northern blots of chicken mRNA. A major band of approximately 0·65–0·85 kb was seen, plus several minor bands of larger molecular weights. Analysis of genomic Southern blots shows that there is one copy of the chicken IGF-I gene.
Alteration in the expression of genes for cholesterol side-chain cleavage enzyme and 21-hydroxylase by hypophysectomy and ACTH administration in the rat adrenal
Authors: Imai T. , Seo H. , Murata Y. , Ohno M. , Satoh Y. , Funahashi H. , Takagi H. , Matsui N.
Published: December 2008

The changes in steady-state levels of mRNA for cholesterol side-chain cleavage cytochrome P-450 (P-450scc) and steroid 21-hydroxylase cytochrome P-450 (P-450c21) caused by hypophysectomy and ACTH treatment were determined in rat adrenals. Hypophysectomy caused marked decreases in adrenal weight and total RNA per gland. Administration of ACTH resulted in increases in adrenal weight and total RNA. A significant correlation between the amount of RNA and adrenal weight was observed. Both P-450scc and P-450c21 mRNAs were decreased by hypophysectomy and increased by ACTH treatment. P-450scc mRNA decreased to 20% and P-450c21 mRNA to 76% of control values 1 day after hypophysectomy. ACTH caused a significant increase in P-450scc mRNA after 3 h. However, a significant increase in P-450c21 mRNA was observed 12 h after administration of ACTH. These results are concordant with previous studies in vitro utilizing cultured adrenocortical cells. Moreover, the induction of steady-state levels of P-450scc mRNA was faster than that observed by other investigators in studies in vitro. These results may indicate that integrity of the adrenal gland in vivo is important for the action of ACTH.
Glucagon-like peptide-1(7–36)amide: characterization of the domain responsible for binding to its receptor on rat insulinoma RINm5F cells
Authors: Gallwitz B. , Schmidt W. E. , Conlon J. M. , Creutzfeldt W.
Published: December 2008

Glucagon-like peptide-1(7–36)amide (GLP-1(7–36)amide) is a potent stimulator of insulin secretion. Receptors for this hormone have been found on different insulinoma-derived cell lines, e.g. the RINm5F cell line which is derived from a radiation-induced rat insulinoma. To characterize the part of the GLP-1(7–36)amide molecule that is responsible for binding to its receptor on RINm5F cells, binding studies with synthetic C-terminal (GLP-1(21–36)amide) and synthetic N-terminal (GLP-1(7–25)) GLP-1 fragments were carried out. GLP-1(21–36)amide showed dose-dependent binding to the GLP-1(7–36)amide receptor but was approximately 1500 times less potent in inhibiting binding of 125I-labelled GLP-1(7–36)amide than the intact hormone. GLP-1(7–25) at concentrations up to 10 μmol/l did not inhibit binding of label. Neither fragment changed intracellular cyclic AMP concentrations, in contrast to GLP-1(7–36)amide which increased intracellular cyclic AMP. GLP-1(21–36)amide, however, acted as a weak partial antagonist of GLP-1(7–36)amide with respect to GLP-1(7–36)amide-dependent stimulation of cyclic AMP production.
Differential effects of β-adrenergic agonists on insulin secretion from pancreatic islets isolated from rat and man
Authors: Lacey R. J. , Berrow N. S. , London N. J. M. , Lake S. P. , James R. F. , Scarpello J. H. B. , Morgan N. G.
Published: December 2008

The selective β2-adrenergic agonist clenbuterol was ineffective as a stimulus for insulin secretion when isolated rat pancreatic islets were incubated with glucose at concentrations between 4 and 20 mM. Inclusion of the phosphodiesterase inhibitor 3-isobutyl-1-methylxanthine led to potentiation of glucose-induced insulin secretion, but did not facilitate stimulation by clenbuterol. Furthermore, maintenance of isolated rat islets for up to 3 days in tissue culture also failed to result in the appearance of a secretory response to β-agonists. By contrast, clenbuterol induced a dose-dependent increase in insulin release from isolated human islets incubated with 20 mm glucose. Clenbuterol did not increase the basal rate of insulin secretion (4 mm glucose) in human islets. Under perifusion conditions, the secretory response of human islets to clenbuterol was rapid, of similar magnitude to that seen under static incubation conditions and could be sustained for at least 30 min. The increase in insulin secretion induced by clenbuterol was inhibited by propranolol, indicating that the response was mediated by activation of β-receptors. In support of this, a similar enhancement of glucose-induced insulin secretion was elicited by a different β2-agonist, salbutamol, in human islets. The results indicate that the B cells of isolated rat islets are unresponsive to β-agonists, whereas those of human islets are equipped with functional β-receptors which can directly influence the rate of insulin secretion.
Secretory patterns of 1α-hydroxycorticosterone in the isolated perifused interrenal gland of the dogfish, Scyliorhinus canicula
Authors: O'Toole L. B. , Armour K.J. , Decourt C. , Hazon N. , Lahlou B. , Henderson I. W.
Published: December 2008

An isolated in-vitro perifused interrenal gland preparation from the dogfish Scyliorhinus canicula was used to study production of quantitatively the major corticosteroid 1α-hydroxycorticosterone (1α-OH-B), measured by radioimmunoassay. Basal secretory rates were 877·1 ± 145 (s.e.m.) fmol/mg per 15 min (n=14) and the preparation remained viable for up to 22 h, as reflected in a brisk response to 10 μm cyclic AMP (cAMP) after this time. Steroid production responded in a dose-dependent manner to porcine ACTH, with 10 μm producing a maximum stimulation of 225% above the basal secretory rate. cAMP (10 μm) produced an increase of 278% above basal, while 1 μm forskolin increased basal secretory rates by 127%. [Val5]- and [Ile5]-angiotensin II (0·1 μm) increased 1α-OH-B production by 120 and 372% respectively over basal secretory rates. Increasing the concentration of K+ in the perfusate from 8 mm to 12, 18, 28 and 40 mm produced a significant rise only at 28 mm. Alterations in the concentration of Na+ and osmolarity of the perifusion medium had inconsistent effects on steroid production. Increased concentrations of urea (from 360 to 720 mm) increased the basal secretory rate by 121%, whilst reducing the concentration of urea (from 360 to 90 mm) had no effect.
Production and utilization of monoclonal antibodies to human/rat corticotrophin-releasing factor-41
Authors: Milton N. G. N. , Hillhouse E. W. , Nicholson S. A. , Self C. H. , McGregor A. M.
Published: December 2008

Murine monoclonal antibodies against human/rat corticotrophin-releasing factor-41 (CRF-41) were produced and characterized for use in the immunological and biological characterization of CRF-41. Spleen cells from BALB/c mice immunized with CRF-41 conjugated to bovine γ-globulin were fused with a BALB/c-derived non-secretor X-63 myeloma line. Hybridomas were selected for CRF antibody production by enzyme-linked immunosorbent assay, and positive hybridomas cloned twice. Three monoclonal antibodies were obtained (KCHMB001, KCHMB002 and KCHMB003) and characterized as IgG1, IgG1 and IgG2a isotypes respectively, with affinity constants for rat CRF-41 of 30, 53 and 34 nmol/l respectively. All three monoclonal antibodies recognize an epitope contained between residues 34 and 41 of the human/rat sequence. The antibodies were able to neutralize the ACTH-releasing activity of rat CRF-41, applied to rat pituitary fragments in vitro, in a dose-dependent manner. Isoelectric focusing showed that KCHMB 003 detected bands of synthetic rat CRF-41 and rat [Met(O)21,38]-CRF-41 at pH 7·1 and 6·8 respectively. Use of KCHMB003 in a two-site enzyme-amplified immunoassay showed that this antibody recognizes both synthetic rat CRF-41 and immunoreactive CRF-41 in rat hypothalamic tissue extracts.
Relaxin gene expression in the porcine follicle during preovulatory development induced by gonadotrophins
Authors: Bagnell C. A. , Tsark W. , Tashima L. , Downey B. R. , Tsang B. K. , Ainsworth L.
Published: December 2008

Northern analysis was used to identify relaxin gene expression in ovaries of prepubertal pigs primed with pregnant mare's serum gonadotrophin (PMSG) and human chorionic gonadotrophin (hCG). The cellular distribution of relaxin transcript in the developing follicle was localized by in-situ hybridization histochemistry. Three probes complementary to non-overlapping regions of the porcine prorelaxin molecule were used to identify relaxin gene expression in ovarian follicular tissue collected 0, 48, 60, 72 and 84 h after treatment with PMSG/hCG. A 1 kb transcript was detected in ovarian extracts of prepubertal gilts from 48 to 84 h after PMSG stimulation. This corresponds to the molecular size of the relaxin transcript reported in the pregnant sow ovary. Relaxin mRNA levels increased in ovaries from animals 48 through 84 h after PMSG. In-situ hybridization showed that the site of relaxin synthesis was the theca interna layer of the developing follicle. Relaxin mRNA was not observed in other follicular cell types, in small or atretic follicles or in follicles from unstimulated animals. The distribution and relative concentration of relaxin mRNA showed a good correlation with in-vitro production and immunohistochemical localization of relaxin previously reported in the developing pig follicle. The presence of both protein and mRNA for relaxin in the growing follicle supports a role for relaxin as a local regulator of ovarian function.
Growth hormone receptors in hypothalamic and extra-hypothalamic tissues
Authors: Fraser R. A. , Attardo D. , Harvey S.
Published: December 2008

Central GH receptors (GHR) have been identified in hypothalamic and extra-hypothalamic tissues of rabbit and chicken brains. Plasma membranes of the rabbit brain demonstrated specific saturable high-affinity, low-capacity binding sites for 125I-labelled GH. RNA extracted from hypothalamic and extra-hypothalamic tissues of rabbit and chicken brains contained mRNA that hybridized with a cDNA probe for the rabbit liver GHR. This transcript was of a similar size to the major GHR mRNA moiety in rabbit liver. The expression of these moieties was age related, and higher in adult than in neonatal animals.
Molecular cloning and nucleotide sequence analysis of complementary DNA for bullfrog prolactin
Authors: Takahashi N. , Yoshihama K. , Kikuyama S. , Yamamoto K. , Wakabayashi K. , Kato Y.
Published: December 2008

A prolactin cDNA was cloned from a cDNA expression library constructed from total RNA of bullfrog (Rana catesbeiana) adenohypophyses by immunoscreening with antiserum against bullfrog prolactin. The cDNA clone thus obtained contained a 249 bp insert. Using this clone as a probe, plaque hybridizations were performed and two additional clones obtained. These clones had a polyadenylation site different from that of the first obtained clone, suggesting that the 3′-untranslated sequence was heterogeneous in length. The longest clone contained 830 bp, which encoded part of the signal peptide and the entire sequence of mature prolactin. The deduced amino acid sequence was in good accord with that determined by direct protein sequencing of purified bullfrog prolactin. The length of the bullfrog prolactin mRNA was estimated by Northern blot analysis to be about 1·0 kb. Homologies of prolactin nucleotide and amino acid sequences between bullfrog and other vertebrates were 64 and 65% for man, 66 and 68% for pig, 61 and 52% for rat, 69 and 74% for chicken, and 50 and 35% for salmon respectively. Highly conserved regions reported for mammalian prolactins also existed in bullfrog prolactin. Homologies of nucleotide and amino acid sequences between prolactin and GH of bullfrog origin were 49 and 25% respectively. Using the cDNA, the content of prolactin mRNA in the pituitary glands of metamorphosing tadpoles was measured. Prolactin mRNA levels rose at the mid-climax stage, suggesting that the increase in plasma and pituitary prolactin levels known to occur at the climax stage accompanies the increase in prolactin synthesis.
A marsupial β-lactoglobulin gene: characterization and prolactin-dependent expression
Authors: Collet C. , Joseph R. , Nicholas K.
Published: December 2008

Analysis of the tammar wallaby β-lactoglobulin cDNA and inferred amino acid sequences reveal extensive sequence divergence from the eutherian β-lactoglobulins. Conserved residues include the cysteines and a number of individual amino acids involved in structure and ligand-binding. The only region of extended similarity is a heptapeptide sequence which may impart specificity to its interaction with a receptor protein. Northern analysis of total mammary RNA revealed two transcripts which result from differential utilization of polyadenylation signals. The concentration of β-lactoglobulin mRNA increased in late lactation and correlates with increases in milk production and levels of milk fat. Quantification of β-lactoglobulin mRNA levels in hormone-stimulated mammary gland explants from tammars in late pregnancy suggests that maximal induction of the gene is dependent on prolactin alone and that expression is not modulated by other hormones known to play a role in the initiation of lactation in eutherians.
The ovine insulin-like growth factor-I gene: characterization, expression and identification of a putative promoter
Authors: Dickson M. C. , Saunders J. C. , Gilmour R. S.
Published: December 2008

Genomic DNA encoding the ovine insulin-like growth factor-I (IGF-I) gene was cloned and sequenced. The predicted amino acid sequence of the mature form of ovine IGF-I was highly homologous to that of human, rat and mouse. Analysis of the DNA sequence between exons 1 and 2 suggested the existence of an alternative 5′ exon (exon 1A) and this was confirmed by polymerase chain reaction (PCR) analysis of sheep liver mRNA. Primer extension of mRNA from exon 1A indicated a class of transcripts which initiated at a point 32 nucleotides 5′ to the Met codon of exon 1A to give a mRNA comprising exons 1A, 2, 3 and 5. In liver these transcripts co-existed with the alternative exon 1, 2, 3 and 5 mRNA form. Analysis by PCR of the 3′ terminus of liver RNA indicated heterogeneity arising from multiple polyadenylation sites; however, of the two possible alternatively spliced 3′ exons, only exon 5 could be detected.

Expression of IGF-I mRNA, as measured by a solution hybridization/RNase protection assay, predominated in the liver of the neonate and the late-gestation fetus; however, lower levels of expression were seen in multiple tissues throughout fetal and neonatal development.
Nutrition and somatomedin XXII: Molecular regulation of insulin-like growth factor-I during fasting and refeeding in rats
Authors: Goldstein S. , Harp J. B. , Phillips L. S.
Published: December 2008

The liver is thought to be the locus of nutritional/hormonal regulation of circulating insulin-like growth factor-I (IGF-I). To probe the basis of nutritional regulation, we examined changes in serum IGF-I, hepatic content of extractable IGF-I immunoreactivity (a high Mr putative precursor) and hepatic IGF-I mRNA during fasting and refeeding in rats.

Preliminary studies revealed that the hepatic level of IGF-I mRNA was consistently reduced only after food was withheld for 3 days, so the effects of refeeding were subsequently examined in such animals. After 3 days of fasting, animals lost 30% of their initial weight; weight regain was apparent within 3 h of refeeding ad libitum and, after 48 h, weight was comparable to initial fed levels. Fasting reduced levels of serum and extractable hepatic IGF-I to 19 and 26% of control (fed) values respectively (both P<0·005 vs control). There was no change in levels of serum IGF-I over the first 3 h of refeeding, but IGF-I rose above fasted levels at both 9 and 48 h (both P<0·005). Extractable hepatic IGF-I rose more slowly and was still below fasted levels after 9 h of refeeding, and modestly, but not significantly, greater than fasted levels after 48 h. The ratio of serum to hepatic IGF-I was decreased compared with control after 3 days of fasting, but increased after 3 and 9 h of refeeding (P<0·02 at 9 h).

Northern blot analysis of total hepatic RNA revealed four species of IGF-I mRNA 0·8–1·1, 2·0, 4·0 and 7·5 kb in size. Each mRNA species fell to 15–28% of control levels after 3 days of fasting (all P<0·001). There was a prompt increase in each transcript after 3 h of refeeding, and all values were significantly (P<0·05) greater than fasted levels at 9 h but, at 48 h, most species were still below control levels. Levels of mRNA for the cytoskeletal proteins β-actin and cyclophilin also fell with fasting, but were restored more rapidly than IGF-I mRNA, to or above control levels after 3 h of refeeding. The observation that IGF-I expression was decreased at 3 h when β-actin and cyclophilin were normalized suggests specificity of regulation. Despite the temporal incongruity between IGF-I mRNA and serum and hepatic IGF-I, there were highly significant correlations (all P<0·002) between each pair of parameters.

Since refeeding of fasted animals led to a rise in IGF-I mRNA which preceded rises in serum and hepatic IGF-I, our findings are consistent with the hypothesis that nutritional regulation of circulating IGF-I involves modulation at the level of hepatic IGF-I mRNA. The changes in the ratio of hepatic to serum IGF-I during fasting and refeeding indicate that there may also be regulation at the level of hepatic release.