bluuebunny/crossref_metadata_2025_split huggingface dataset:
Identification of high molecular weight peptides in bovine adrenomedullary chromaffin granules using monoclonal antibodies to a preproenkephalin A fusion peptide
Authors: Jackson S. , Spruce B. A. , Glover D. M. , Glynn B. P. , Lowry P. J. 
Published: December 2008

ABSTRACT
Two mouse monoclonal antibodies (PE-1 and PE-2) raised to a β-galactosidase—preproenkephalin A(69– 207) fusion peptide recognize pro-enkephalin A (pro-enk-A) peptides of 33–5 kDa isolated from bovine adrenal chromaffin granules. The preliminary characterization of the high molecular weight adrenomedullary pro-enk-A peptides recognized by PE-1 and PE-2 is described. The high molecular weight peptides were resolved after Sephadex G-50 chromatography and high-performance liquid chromatography (HPLC) into three components (peaks I, II and III). Immunoblot analysis showed each HPLC peak to be heterogeneous. Peak I contained PE-1-and PE-2-immunoreactive peptides of 33, 29, 24 and 22 kDa; peak II contained a peptide of 22 kDa recognized by PE-2, and peptides of 24 and 22 kDa recognized by PE-1; peak III contained a PE-2-immunoreactive peptide of 15 kDa and PE-1-immunoreactive peptide of 18 kDa. Using polyclonal antibodies to peptide F and methionineenkephalin-Arg6-Gly7-Leu8 (MetEnk-RGL), the 22 kDa band cross-reacted with both MetEnk-RGL and peptide F antibodies, whilst the 24 kDa band was shown to possess predominantly MetEnk-RGL immunoreactivity. The 15 kDa (PE-2-immunoreactive) band was recognized by the peptide F but not the MetEnk-RGL antibody, whereas the polyclonal antibodies did not recognize the 18 kDa (PE-1-immunoreactive) band. We propose that the immunological and size characteristics of some of these peptides (29, 24/22, 15 kDa) suggest their similarity to the peptides of predicted molecular mass 23·3, 18·2 and 12·6 kDa previously found in bovine adrenal medulla. The results also indicate the existence of high molecular weight pro-enk-A peptides shortened at the N-terminus. The use of an immunoradiometric assay designed to measure the proenk-A-derived 18·2 kDa peptide using PE-2 and an affinity purified and radioiodinated MetEnk-RGL IgG has supported these findings.
Inhibitory effects of alkane sulphonates on the function of immature rat Leydig, Sertoli and peritubular cells cultured in vitro
Authors: Verhoeven G. , Cailleau J. , Morris I. D. 
Published: December 2008

ABSTRACT
Ethane 1,2-dimethane sulphonate (EDS) selectively destroys Leydig cells in the interstitium of the testis of adult rats. The toxic activity of this compound is much less obvious in the immature rat testis. We examined the effects of EDS, its monomethyl derivative and busulphan on cultured interstitial cells, percoll-purified Leydig cells, Sertoli cells and peritubular cells derived from immature rats. The studies with interstitial cells and Leydig cells showed that EDS (40–160 μg/ml) blocked the conversion of C21 and androgen precursors into testosterone and androstenedione. Higher concentrations of this compound also inhibited the production of C21 steroids and the LH-induced production of cyclic AMP (cAMP). The observed effects required a latent period of at least 8 h and were slowly reversible. Isolated cells were more sensitive to EDS than monolayer cultures. Reaggregation cultures were even less sensitive. EDS was markedly more effective on immature Leydig cells than its monomethyl derivative and busulphan. In cultured Sertoli cells FSH-inducible aromatase activity, cAMP production, androgen-binding protein (ABP) production and the secretion of a paracrine factor with Leydig cell-stimulatory activity were markedly reduced by busulphan. In these cells, busulphan was clearly more active than EDS and its monomethyl derivative. The production of paracrine factors which increase ABP production and decrease FSH-inducible aromatase activity in Sertoli cells was studied as a parameter of the effects of alkane sulphonates on peritubular cells. Only busulphan markedly decreased the production of these paracrine factors. It is concluded that EDS displays a selective toxicity to Leydig cells derived from immature animals and that, apart from its effects on germ cells, busulphan may also directly impair the function of Sertoli cells and peritubular cells.
Pro-opiomelanocortin gene expression in human phaeochromocytomas
Authors: de Keyzer Y. , Rousseau-Merck M.-F. , Luton J.-P. , Girard F. , Kahn A. , Bertagna X. 
Published: December 2008

ABSTRACT
Phaeochromocytoma is an occasional cause of the ectopic ACTH syndrome. The mechanisms of proopiomelanocortin (POMC) gene expression were analysed in 11 human tumours not associated with Cushing's syndrome, by detecting and characterizing the POMC mRNA. A DNA probe corresponding to most of the protein-coding region of the third exon was used in Northern blot studies of total and poly(A)+ RNA. All tumours contained a short (800 bases) mRNA species different from the 1200 base mRNA species of the human pituitary. This short mRNA was also present in the normal adrenal, where S1 mapping showed that it resulted from transcription initiation within the third exon. However, in two tumours, equivalent amounts of the 1200 base mRNA were also present, and in one of them a third POMC mRNA of approximately 1450 bases was detected. These data show that POMC gene expression occurs in all phaeochromocytomas. It is suggested that excess production of the 1200 bases (or the larger, 1450 base) mRNA in some tumours may be responsible for the rare occurrence of the ectopic ACTH syndrome.
Regulation of LH subunit and prolactin mRNA by gonadal hormones in mice
Authors: Saade G. , London D. R. , Lalloz M. R. A. , Clayton R. N. 
Published: December 2008

ABSTRACT
The effect of castration and gonadal steroid replacement on the concentrations of LH-β and α subunit and prolactin mRNA was examined in mice.
Mouse LH-β, α and prolactin mRNAs were approximately 0·8, 0·7 and 1·1 kb in size respectively. After ovariectomy, LH-β mRNA levels increased 2- to 2·5-fold, while α mRNA levels increased 2·5-fold 6 and 10 days after ovariectomy. Serum LH rose after 2 days to reach six times control values at 10 days. Pituitary LH content doubled by 8 days after ovariectomy. Prolactin mRNA levels decreased to 50–60% of control at 3, 6, 8 and 10 days after ovariectomy and parallelled the fall in serum prolaction. Pituitary prolactin content fell more slowly, to 50% of intact control values by 10 days. The increase in both LH-β and α subunit mRNA, and decrease in prolactin mRNA, and serum and pituitary hormone changes, after ovariectomy were prevented by oestradiol or oestradiol plus progesterone replacement.
Levels of LH-β mRNA increased more quickly in male than in female mice, theearliest change being seen 24 h after orchidectomy. Maximum values (two- to threefold) were found on day 6 after orchidectomy. Concentrations of α mRNA increased by 12 h to between 2 and 2·5 times control from 3 to 10 days after orchidectomy. Serum LH doubled by 12 h and was three to five times greater than control values up to 10 days. Pituitary LH content fell by 48 h before gradually increasing to intact values after 10 days. Prolactin mRNA levels decreased progressively from 2 days after orchidectomy, and this decrease was preceded by a fall in serum and pituitary prolactin which remained low throughout the experiment. Testosterone treatment attenuated the rise in α mRNA, prevented the rise in LH-β mRNA and serum LH and partially restored the decrease in prolactin mRNA seen after orchidectomy.
We conclude that in mice, as in rats and ewes, both LH-β and α subunit mRNAs are negatively regulated by gonadal steroids, whereas prolactin mRNA is positively regulated, although there are temporal differences in patterns of mRNA responses between males and females. By comparison with female rats the rise in LH-β mRNA after ovariectomy was slower in mice. Moreover, the discordant changes in pituitary LH content and LH subunit mRNAs seen in mice after castration were not observed in rats. Furthermore, pituitary prolactin and prolactin mRNA do not fall after orchidectomy of rats. The modest (50%) increase of LH-β mRNA after castration of mice suggests that an increase in mRNA is not necessarily required for increased LH production.
Isoprenaline-induced and constitutive members of a proline-rich protein sub-group from mouse parotid glands studied with monoclonal antibody NAL1
Authors: Divecha N. , Mansouri H. , Peat D. , Cope G. , Partridge L. , McDonald C. J. 
Published: December 2008

ABSTRACT
Members of the proline-rich protein (PRP) family of mouse parotid glands were analysed before and after stimulation with the β-agonist isoprenaline by using a monoclonal antibody raised against the induced PRP A30 (GP-27). Antibody NAL1 reacted strongly with isoprenaline-induced B-type PRP precursors and their salivary counterparts, but not against the A-type PRPs A10 (Gp-66) and A20 (GP-45) or human salivary proteins, and it is likely that NAL1 recognizes a proline-rich repeat variant unique to this group of rodent PRPs. PRP-related antigens were observed in the parotid glands (N10 and N20) and saliva of normal mice. The antigens were located immunocytochemically in secretory granules of parotid acinar cells of both normal and isoprenaline-stimulated mice. The total amount of PRP antigens increased 16-fold from 2·5 to 40% of parotid protein after 10 days of isoprenaline treatment, as estimated by enzyme-linked immunosorbent assay. Immunoblotting showed that new PRP species appeared during the period of increase. After treatment with isoprenaline, B-type PRPs appeared first, followed by A30 and another member of the family. These results show that the mouse PRP family is larger than previously thought and can be divided immunologically into sub-groups. That a subset of PRPs are produced in the normal mouse indicates that there is differential β-adrenergic regulation within the family, and also has implications for the role of PRPs in the normal maintenance of healthy dentition and other processes.
Characterization of prostaglandin E2 binding to uterine membranes from baboon, rabbit and tree shrew (Tupaia belangeri)
Authors: Hodam J. R. , Snabes M. C. , Kuehl T. J. , Jones M. A. , Harper M. J. K. 
Published: December 2008

ABSTRACT
Membrane preparations (100 000 g pellet) of rabbit, baboon and tree shrew (Tupaia belangeri) uteri were studied for binding of [3H]prostaglandin E2 ([3H]PGE2). Unbound [3H]PGE2 was separated by filtration through Whatman GF/F filters. Nonspecific binding was determined by the amount of radioactivity associated with the filters in the presence of a 100-fold excess of radioinert PGE2. PGE2 bound to membranes could be displaced by some other prostaglandin (PG) molecules: PGE1, 16,16-dimethyl-PGE2, PGA1 and PGF2α, but not by 6-keto-PGF1α, PGD2 or arachidonic acid. No PGE2 binding was detected using either membrane ghosts from red blood cells or liposomes. Apparent equilibrium of the binding was reached by 60 min. There was no difference in dissociation constant (Kd) values between rabbits of different reproductive stages (mean range±s.e.m. was from 4·6±0·3 to 5·5 ± 1·0 nm), but pregnant baboons showed a significantly lower value (3·3±0·4 nm) than did cyclic animals (12·0 ± 2·0 nm). Binding capacity (Bmax) values, in contrast, were different only between oestrous rabbits and other reproductive stages. The small amounts of [ill] tissue only permitted estimates of the Kd and Bmax values to be made; these were 3·8 nm and 499 fmol/mg protein for oestrous animals and 5·4 nm and 674 fmol/mg protein for animals on day 7 of pregnancy, assuming only one class of sites. The present results demonstrate the presence of specific binding sites for PGE2 in uteri from several species.
Melatonin-binding sites in the rat brain and pituitary mapped by in-vitro autoradiography
Authors: Williams L. M. 
Published: December 2008

ABSTRACT
Using picomolar concentrations of [125I]iodomelatonin and in-vitro autoradiography, specific melatonin-binding sites have been mapped in the rat brain and pituitary. Using this same technique, high-affinity melatonin receptors had previously been identified in the suprachiasmatic nucleus (SCN) and median eminence regions of the rat hypothalamus. The presence of melatonin binding in the SCN has been confirmed, but the second area of binding has been identified as the pars tuberalis of the pituitary, and a completely novel area of binding is also reported in the area postrema. The existence of lower affinity melatonin receptors in the rat brain was also investigated using in-vitro autoradiography and higher concentrations of [125I]iodomelatonin. No further sites of specific binding were, however, disclosed.
A major C-peptide deletion prevents secretion of a mutant human proinsulin from transfected monkey kidney cells
Authors: Shakur Y. , Shennan K. I. J. , Taylor N. A. , Docherty K. 
Published: December 2008

ABSTRACT
The biosynthesis and secretion of human proinsulin and a mutant human proinsulin with a major deletion in the C-peptide, (des 38–62)proinsulin, was studied in monkey kidney cells (Cos-7) transfected with cDNAs encoding the respective normal or mutant human preproinsulins. Transfected cells were labelled with [3H]leucine, and insulin-like material was immunoprecipitated and analysed by sodium dodecyl sulphate-polyacrylamide gel electrophoresis. It was found that the prepeptide was removed from both the normal and mutant preproinsulins, and that there was no further processing to insulin. The normal proinsulin was rapidly released from the transfected cells, with little intracellular accumulation, while the mutant proinsulin was retained within the cell, with only small quantities of radiolabelled material in the medium. The intracellular mutant proinsulin was membrane bound and located predominantly within a microsomal fraction. These results suggest that C-peptide plays an important role in the efficient transfer of proinsulin through the early stages of the secretory pathway.
Characterization of relaxin-stimulated cyclic AMP in cultured rat anterior pituitary cells: influence of dopamine, somatostatin and gender
Authors: Cronin M. J. , Malaska T. 
Published: December 2008

ABSTRACT
Relaxin is a hormone associated with pregnancy that relaxes uterine smooth muscle and softens the connective tissues of the cervix and pelvis. In spite of these well-characterized tissue responses, the second messenger system linked to the relaxin receptor and the range of target tissues are only modestly understood. We found that relaxin enhanced the cyclic AMP levels in anterior pituitary cells from adult female rats. Relaxin induced a maximal 5·7±0·5-fold (mean±s.e.m.) stimulation of cyclic AMP accumulation and had an excitatory concentration for half maximal effect (EC50) of 0·4±0·1 nm, while human relaxin A and B chains had no such activity (EC50> 1 μm). Pertussis toxin amplified the efficacy of relaxin by 1·5±0·1-fold, indicating the intervention of a G coupling protein. The response to relaxin was reversible with washing, and desensitized slowly with continuous exposure to relaxin.
In an attempt to define the physiological role for relaxin at the anterior pituitary, we found that two of the major hypophysiotrophic hormones of the brain (dopamine and somatostatin) markedly inhibited the relaxin stimulation of cyclic AMP. There was also a significant correlation of the response magnitude with the gender of the donor rat. Anterior pituitary cells from adult males exhibited a mean twofold maximal stimulation after relaxin, compared with the sixfold increase measured in cells from female rats. We hypothesize a novel physiological function of relaxin, that of signalling the feminine anterior pituitary.
Metabolism of 25-hydroxyvitamin D3 to 24,25-dihydroxyvitamin D3 and its sensitivity to ketoconazole in 1α,25-dihydroxyvitamin D3-differentiated HL60 cells
Authors: Hayes M. E. , Bayley D. , Mawer E. B. 
Published: December 2008

ABSTRACT
Regulation of the metabolism of [3H]25-hydroxyvitamin D3 ([3H]25-(OH)D3) in vitro to material with the characteristics of [3H]24,25-dihydroxyvitamin D3 ([3H]24,25-(OH)2D3) has been studied in the human promyelocytic cell line HL60. Synthesis of 24,25-(OH)2D3 was induced in a dose-dependent manner in cells pretreated with 0·1–100 nm 1α,25-dihydroxyvitamin D3 (1α,25-(OH)2D3) for 4 days. This treatment also inhibited cell proliferation and stimulated differentiation to a macrophage phenotype that was characterized by staining for non-specific esterase (NSE) activity. The ability to synthesize [3H]24,25-(OH)2D3 from [3H]25-(OH)D3 and the expression of NSE activity both responded to changes in concentration of 1α,25-(OH)2D3 in the culture medium in a parallel manner. Synthesis of [3H]24,25-(OH)2D3 was linear when the incubation time was between 1 and 8 h and the cell number between 1 and 12×106 cells/incubation. The optimum substrate concentration for its synthesis was 125 nm, giving an apparent Michaelis constant of 360 nm. The identity of the [3H]24,25-(OH)2D3 synthesized by these cells was confirmed by co-chromatography with authentic 24,25-(OH)2D3 on normal-phase and reverse-phase high-performance liquid chromatography systems and by its reaction to sodium-m-periodate. Cells that had been exposed to 100 nm 1α,25-(OH)2D3 for 4 days synthesized 2·17±0·07 (s.e.m.) pmol 24,25-(OH)2D3/106 cells per h. This synthesis was inhibited in a dose-dependent manner over a concentration range of 0·01–1 μm by the drug ketoconazole, an antimycotic imidazole which is a known inhibitor of certain cytochrome P-450 enzyme systems, suggesting that the HL60 25-(OH)D3-24-hydroxylase is also a P-450-dependent enzyme system.
Both α- and β-calcitonin gene-related peptides are present in plasma, cerebrospinal fluid and spinal cord in man
Authors: Wimalawansa S. J. , Morris H. R. , Maclntyre I. 
Published: December 2008

ABSTRACT
The presence of both α- and β-calcitonin gene-related peptide (CGRP) was demonstrated by oxidation and by CNBr cleavage in extracts of plasma, cerebrospinal fluid and spinal cord in man. This was achieved by the use of both CNBr cleavage and oxidation of the methionine residue present in the human β-CGRP molecule. This study demonstrates that around 50% of CGRP immunoreactivity in plasma, cerebrospinal fluid and spinal cord is not α-CGRP, but corresponds to β-CGRP-like activity. Furthermore, experiments with CNBr also suggest the presence of another methionine-containing CGRP-like peptide in all three extracts.
Differential regulation of calbindin-D28k mRNA in the intestine and eggshell gland of the laying hen
Authors: Bar A. , Striem S. , Mayel-Afshar S. , Lawson D. E. M. 
Published: December 2008

ABSTRACT
The effect of shell calcification and 1,25-dihydroxyvitamin D3 (1,25-(OH)2D3) on calbindin-D28k (previously known as vitamin D-dependent calcium-binding protein) and calbindin mRNA was investigated in the intestine and eggshell gland (ESG) of juvenile female chicks, laying hens and non-laying female birds with active gonads. Increasing amounts of 1,25-(OH)2D3 were fed to laying hens and juvenile birds treated with oestradiol to develop the ESG. The intestinal concentration of calbindin was increased 30-fold by 1,25-(OH)2D3 in chicks treated with oestradiol and fed a vitamin D-deficient diet. In these same animals, 1,25-(OH)2D3 had no effect on the formation of calbindin mRNA or calbindin in the ESG even though fully viable 1,25-(OH)2D3 receptors are present in this tissue. In laying birds fed adequate amounts of vitamin D3, intestinal, but not ESG, calbindin was increased by the addition of 1,25-(OH)2D3 to the diet. At the onset of egg production the concentrations of calbindin and calbindin mRNA were increased in the intestine and ESG. This increase occurred within the period of calcification of the first egg, through a process unaffected by vitamin D. Calcification of the first egg increased the concentration of calbindin in the ESG by eight- to tenfold, although the concentration of calbindin mRNA was increased by only two- to threefold.
These results suggest that the induction of calbindin synthesis by 1,25-(OH)2D3 or by the egg calcification process is associated with an increase in the concentration of calbindin mRNA in the ESG and intestine. They also suggest that the vitamin D-dependent physiological changes in the ESG occurring during the calcification process do not include an increase in calbindin synthesis, that calbindin is induced and/or regulated by a mechanism requiring additional factors besides 1,25-(OH)2D3, and that post-transcriptional regulation of calbindin synthesis in these tissues may be possible.
Comparison of the pretranslational regulation of FSH synthesis by gonadal steroids in rats and mice
Authors: Rodin D. A. , Abbot S. D. , Saade G. , Clayton R. N. 
Published: December 2008

ABSTRACT
There are significant differences between rats and mice in the gonadal regulation of several aspects of gonadotroph function. To investigate whether these extend to the pretranslational regulation of FSH synthesis by gonadal steroids, we have measured FSH-β mRNA levels following gonadectomy and sex-steroid replacement and have related these to serum and pituitary FSH as a reflection of overall hormone synthesis.
In ovariectomized rats, FSH-β mRNA levels increased by 8 h, decreased, and then rose progressively over the next 28 days. A similar pattern of response was observed in orchidectomized rats. In mice, there were progressive increases in FSH-β mRNA levels in both males and females following gonadectomy, without evidence of the early peaks observed in rats. In both species, the change in FSH-β mRNA levels after gonadectomy was greater in females than in males. These changes in FSH-β mRNA following gonadectomy were paralleled by changes in the serum FSH concentration. In ovariectomized female rats and mice, pituitary FSH stores increased by 8 h and 3 days respectively, whereas in male rats, pituitary FSH content did not rise until 10 days after orchidectomy. The most striking species difference was the marked and prolonged reduction of pituitary FSH after orchidectomy of mice.
Treatment of rats and mice from the time of ovariectomy, with a dose of oestradiol that prevents increases in serum LH, only partially attenuated the rises in FSH-β mRNA and serum FSH and did not prevent the increase in pituitary FSH content. Treatment of intact or orchidectomized rats with testosterone suppressed FSH-β mRNA levels to 50% below intact control values without affecting pituitary FSH content. In mice, testosterone treatment for 10 days reduced the post-castration increase in FSH-β mRNA by only 26%, and prevented the fall in pituitary FSH content, although the increased serum concentration of FSH was unaffected.
In conclusion: (1) there is a good correlation between FSH-β mRNA levels and overall FSH biosynthesis in male and female rats and female mice, but this relationship is less obvious in male mice where pituitary FSH stores are not increased; (2) the inability of oestradiol to prevent completely the post-ovariectomy increase in FSH-β mRNA and FSH synthesis in female rats and mice indicates either that other gonadal products are necessary or that higher doses of oestradiol are required than for complete suppression of LH synthesis; (3) whilst the post-gonadectomy increases in FSH-β mRNA are larger in the female of both species, there are no major differences between rats and mice in the regulation of FSH-β gene expression by sex steroids.