bluuebunny/crossref_metadata_2025 huggingface dataset:
Multiple forms of parathyroid hormone-like proteins in a human tumour
Authors: Docherty H. M. , Heath D. A.
Published: December 2008

The extensive chromatographic characterization of four parathyroid hormone (PTH)-like proteins in a human bronchial carcinoid tumour associated with humoral hypercalcaemia and severe osteitis fibrosa is described. PTH-like bioactivity was detected in acetic acid extracts of the tumour using an in-vitro osteosarcoma cell bioassay. The active tumour proteins were positively charged at physiological pH and had apparent Mr of approximately 29 000, 16 000, 4000–9000 and < 4000. The proteins were immunologically distinct from PTH, but each stimulated PTH-sensitive adenylate cyclase in cultured osteoblastic cells. There was no evidence of PTH gene expression by the tumour. These proteins represent different molecular forms of PTH-related protein.
Immunological distribution of one form of insulin-like growth factor (IGF)-binding protein and IGF peptides in human fetal tissues
Authors: Hill D.J. , Clemmons D. R. , Wilson S. , Han V.K.M. , Strain A.J. , Milner R.D.G.
Published: December 2008

Insulin-like growth factors (IGFs) are expressed by, and are biologically active on, human fetal cells. The mitogenic actions of IGF-I are modulated by the 21–41 kDa class of IGF-binding proteins (IGF-BPs). Using a rabbit anti-human IGF-BP antibody raised against a highly pure 26 kDa IGF-BP derived from amniotic fluid, we have compared the cellular location of IGF-BP and IGF peptides in tissue sections from prostaglandin-induced human abortuses of 14–16 weeks of gestation. The monoclonal and polyclonal antibodies used were raised against human IGF-I, but did not distinguish between IGF-I and IGF-II. Positive staining for IGF-BP was seen in every tissue except brain, spleen and thyroid. With the exception of skin, the cellular distribution of IGF-BP was similar to that of IGF peptides. Strong immunostaining was found in hepatocytes, hepatic erythropoietic cells, pulmonary epithelium, the tubular epithelium of kidney, intestinal epithelia, the fetal adrenal cortex and cardiac and skeletal muscle fibres. In skin, IGF-BP was located throughout the dermis and in the germinal layer of the epidermis. IGF peptide in skin was restricted to the deeper dermal layers. In the tibial epiphyseal growth plate both IGF-BP and IGF peptide were located in chondrocytes throughout the proliferation and hypertrophic zones. The similarity in distribution of IGF-BP and IGF peptides in fetal tissues suggests that the latter may exist predominantly complexed to IGF-BP in or on the surfaces of cells in vivo. The distribution of IGF-BP may define the sites of biological action of IGF peptides.
Multiple forms of 17β-hydroxysteroid oxidoreductase in human breast tissue
Authors: Tait G.H. , Newton C.J. , Reed M.J. , James V.H.T.
Published: December 2008

17β-Hydroxysteroid oxidoreductase, the enzyme that catalyses the interconversion of oestradiol and oestrone, is known to be present in human breast tissue. However, it is not known whether one or more forms of the enzyme is present. Homogenates of breast adipose tissue and breast glandular tissue were fractionated and fractions assayed in the oxidative direction with NAD+ and NADP+ as coenzymes, and in the reductive direction with NADH and NADPH as coenzymes. Ultracentrifugation of homogenates showed that there was membrane-bound activity and soluble activity. The soluble activity was due to a number of forms of the enzyme with different molecular weights, three in breast adipose tissue and two in breast glandular tissue, as shown by fractionation with (NH4)2SO4 followed by chromatography on Sephadex G-200. The forms of the enzyme isolated differed in their affinities for substrates and coenzymes and in the relative rates at which they catalysed the oxidative and reductive reactions. Preliminary experiments with breast tumours showed that they also contained membrane-bound activity and more than one soluble form of the enzyme.
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

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

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

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

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

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

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

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

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

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

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

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

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

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.
Somatostatin binding to chicken adenohypophysial membranes
Authors: Harvey S. , Attardo D. , Baidwan J. S.
Published: December 2008

[125I-Tyr1]-Somatostatin (SRIF)-binding sites were demonstrated on crude plasma membrane preparations from chicken pituitary glands. These binding sites were saturable and of high affinity (dissociation constant <1·0 nm) and low capacity (maximal binding capacity <200 fmol/mg protein) and were specific for SRIF moieties. The number and affinity of these binding sites in the caudal lobe of the pituitary, in which somatotrophs predominate, were similar to those in the cephalic lobe, in which lactotrophs and thyrotrophs are confined. Gonadotrophs are present in the caudal lobe, but whereas exogenous SRIF inhibited secretagogue-induced GH release from incubated pituitary glands, it had no effect on basal or secretagogue-induced LH release. The half-maximal binding of SRIF to the caudal lobe membranes (3 nm) was similar to that required for half-maximal suppression of TRH-induced GH release, suggesting a role for these binding sites in the regulation of GH secretion in birds.
Differential expression of inhibin α and βA subunit genes in rat and mouse ovarian follicles during pregnancy
Authors: Penschow J. D. , Aldred G. P. , Darling P. A. , Haralambidis J. , Hammond V. E. , van Leeuwen B. H. , Mason A. J. , Niall H. D. , Seeburg P. , Coghlan J. P.
Published: December 2008

Relative levels of rat ovarian α inhibin (αI) and βA inhibin (βAI) mRNAs were measured during pregnancy by dot-blot hybridization of ovarian poly(A+) RNA. Follicular patterns of αI and βAI expression in contralateral ovaries from the same rats were also studied by hybridization histochemistry. Oligodeoxynucleotide probes specific for porcine αI and βAI were synthesized, 32P end-labelled and used as hybridization probes on dot-blots of ovarian RNA and frozen sections of ovarian tissue from pregnant rats. During pregnancy, levels of αI and βAI mRNAs remained fairly constant from day 7 after mating until parturition and then fell within 16 h post partum. In all ovaries observed, expression of inhibin genes was located in granulosa cells of healthy antral follicles. In general, the strongest signals for αI and βAI mRNAs were obtained in large follicles, with weaker signals in smaller follicles. Follicular patterns of αI and βAI expression during pregnancy were often dissimilar when βI and βAI were compared over a range of follicles. Considerable βI mRNA was detectable in some follicles in which βAI was reduced or undetectable, despite strong signals for both αI and βAI in an adjacent follicle. Essentially, αI mRNA levels were relatively consistent between groups of follicles, whereas βAI levels varied considerably. βAI mRNA was never observed in a follicle in the absence of αI mRNA, indicating that activin production in any follicle occurs in the presence of αI mRNA. Similar patterns of expression were observed in ovaries from pregnant mice. We have shown that expression of αI and βAI inhibin genes is not regulated uniformly within follicles of pregnant rat and mouse ovaries.
Structure of an interferon-α2 gene expressed in the bovine conceptus early in gestation
Authors: Stewart H. J. , McCann S. H. E. , Flint A. P. F.
Published: December 2008

Genomic and cDNA clones for bovine trophoblast interferon (IFN) have been isolated by probing a bovine genomic library and a bovine embryonic (day-18 post coitus) cDNA library respectively with the ovine trophoblast IFN cDNA. The two DNA sequences were identical; sequence analysis demonstrated 80% identity between the amino acid sequence of bovine trophoblast IFN and ovine trophoblast IFN, and 70% identity with a previously identified bovine IFN-α2. Southern blotting of bovine genomic DNA indicated the presence of a minimum of three trophoblast IFN genes. Primer extension analysis identified the transcription start site in the 5′ flanking region of the bovine IFN gene. Computer-aided analysis of the 5′ flanking sequence demonstrated a similarity with that of bovine IFN-α2 and the existence of a possible viral induction sequence.
Cyclic AMP and calcium in mouse mammary tumour virus expression: effects and post-transcriptional site of action
Authors: Bolander F. F. , Blackstone M. E. , Bradham B. M.
Published: December 2008

The role of cyclic AMP (cAMP), calcium, calmodulin and protein kinase C (PKC) in the expression of both mouse mammary tumour virus (MMTV) RNA and an MMTV glycoprotein, gp58, was investigated in normal mammary epithelium in culture. None of these second messengers had any effect on MMTV RNA. Dibutyryl cAMP alone had no effect on gp58 levels but, at low concentrations (0·05–0·1 mm), it nearly doubled the induction seen with insulin, cortisol and prolactin; higher concentrations were inhibitory. Although a calcium ionophore (A23187), either alone or with hormones, was ineffective, a calcium channel blocker (verapamil) reduced hormonal induction of gp58 by 80%, and a calmodulin inhibitor (W-13) reduced it by 90%. Two PKC activators, a phorbol ester and a diacylglyceride, were ineffective alone, with hormones or with the calcium ionophore. The following conclusions can be made: (1) cAMP, calcium and calmodulin play an important role in MMTV expression, (2) these second messengers all act post-transcriptionally, since they do not affect MMTV RNA, and (3) PKC does not appear to have a role in MMTV production in normal mammary epithelium.
Adenosine and its analogue ( − )-N6-R-phenyl-isopropyladenosine modulate anterior pituitary adenylate cyclase activity and prolactin secretion in the rat
Authors: Schettini G. , Landolfi E. , Meucci O. , Florio T. , Grimaldi M. , Ventra C. , Marino A.
Published: December 2008

The effect of adenosine and its analogue ( − )-N6-R-phenylisopropyladenosine (PIA) on both anterior pituitary adenylate cyclase activity and prolactin secretion was examined in the rat. Adenosine inhibited basal adenylate cyclase activity in a dose-dependent manner and also reduced the stimulation of the enzyme by vasoactive intestinal peptide (VIP). Likewise, in primary cultures of anterior pituitary cells, adenosine decreased prolactin secretion in both basal and VIP-stimulated conditions. In perifusion experiments, adenosine also inhibited prolactin release in both basal and TRH-stimulated conditions. PIA produced a biphasic pattern of response of basal adenylate cyclase activity, being inhibitory at low and stimulatory at high concentrations. In VIP-stimulated conditions, low concentrations of PIA inhibited both adenylate cyclase activity and prolactin release from primary cultures of pituitary cells, while no additive stimulatory effect was seen at high concentrations. Similarly, low concentrations of PIA reduced both basal and TRH-stimulated prolactin release from perifused pituitaries, while increasing PIA concentrations restored prolactin release. These data show that adenosine affects basal and stimulated prolactin secretion from anterior pituitary cells. Adenosine receptors seem to be coupled to the adenylate cyclase system in the anterior pituitary gland, suggesting a possible relationship between the effect of adenosine on adenylate cyclase activity and prolactin secretion.
Trypsin stimulation of aldosterone and 18-hydroxycorticosterone production by rat adrenal zona glomerulosa tissue is mediated by activation of protein kinase C
Authors: Vinson G. P. , Laird S. M. , Hinson J. P. , Mallick N. , Marsigliante S. , Teja R.
Published: December 2008

When rat adrenal whole capsules, containing the zona glomerulosa, were incubated, addition of the protein kinase C inhibitors TMB-8 (10 μmol/l), W7, H7, polymyxin-B and sphingosine (all 1 μmol/l) was found to inhibit the steroidogenic response to trypsin. Aldosterone and 18-hydroxycorticosterone were strongly, and corticosterone moderately, affected, while the production of 18-hydroxydeoxycorticosterone was neither stimulated by trypsin nor inhibited by the protein kinase C inhibitors. Addition of neomycin, which prevents substrate interaction with phospholipase C, also inhibited the response to trypsin, while addition of phospholipase C itself stimulated aldosterone, 18-hydroxycorticosterone and corticosterone production with the same tissue sensitivity as trypsin. Addition of phospholipase A2 had no effect. Direct assay of protein kinase C activity showed that trypsin stimulation effected the translocation of Ca2+/phospholipid-activated protein kinase C from the cytosolic to the membrane fraction. When glomerulosa tissue was incubated with [32P]ATP, and cytosolic proteins were subjected to isoelectric focusing on polyacrylimide gels, autoradiography showed that incorporation of 32P into several protein components was increased by trypsin stimulation.

It was concluded that trypsin exerts its stimulatory effects on steroidogenesis by activating protein kinase C; not, however, by generating the Ca2+/phospholipid-independent fragment, but possibly by enhancing the activity of phospholipase C.
Site-specific mutagenesis of human chorionic gonadotrophin (hCG)-β subunit: influence of mutation on hCG production
Authors: Azuma C. , Miyai K. , Saji F. , Kamiura S. , Tokugawa Y. , Kimura T. , Ohashi K. , Koyama M. , Iijima Y. , Kashiwai T. , Hayashizaki Y. , Tanizawa O.
Published: December 2008

The heterodimeric glycoprotein hormones, human chorionic gonadotrophin (hCG), LH, TSH and FSH, consist of two non-covalently linked subunits, the α and β subunits. The β subunit is specific for each hormone and is responsible for the biological specificity, but the β subunits of different hormones show some degree of structural homology. The CAGY (cysteine-alanine-glycine-tyrosine) region is one of the amino acid sequences that is homologous in different β subunits and is highly conserved between species. In the present study, site-specific in-vitro mutagenesis was used to change three individual nucleotides in the centre of the CAGY region of the hCG-β subunit, and the effects of these mutations on hCG production was determined by in-vitro transcription and then translation in Xenopus laevis oocytes. The results indicate that the CAGY region, particularly the glycine residue at position 36 in the β subunit, is essential for the production of hCG. This finding is consistent with previous studies showing that this region is necessary for the biological activity of human TSH.
Steroid hormones induce macrophage colony-stimulating factor (MCSF) and MCSF receptor mRNAs in the human endometrium
Authors: Azuma C. , Saji F. , Kimura T. , Tokugawa Y. , Takemura M. , Samejima Y. , Tanizawa O.
Published: December 2008

We investigated the biological effects of sex-steroid hormones, secreted from the corpus luteum and placenta, on the induction of mRNAs encoding macrophage colony-stimulating factor (MCSF) and c-fms proto-oncogene (MCSF receptor) in human endometrium. RNA was extracted from the placenta and endometrium of both pregnant and non-pregnant women, and Northern blot analysis was performed on poly(A)+ RNA using MCSF or c-fms proto-oncogene cDNA as the probe. Results showed: (1) that MCSF mRNA was expressed in the placenta and endometrium of the pregnant uterus, (2) that c-fms proto-oncogene mRNA was also expressed in the placenta and endometrium of the pregnant uterus, and (3) that exogenous sex-steroid hormones could induce the expression of MCSF and c-fms proto-oncogene mRNAs in the endometrium of non-pregnant women. These results indicate that sex-steroid hormones secreted by the corpus luteum and/or placenta influence endometrial and placental growth and differentiation via a mechanism of action involving local production of MCSF and its receptor.
Thyrotrophin-releasing hormone stimulates polyphosphoinositide metabolism in the frog neurointermediate lobe
Authors: Desrues L. , Tonon M. C. , Vaudry H.
Published: December 2008

Previous studies have demonstrated that TRH is a potent stimulator of α-MSH secretion from frog pituitary melanotrophs. In order to determine the intracellular events responsible for TRH-evoked α-MSH release, we have investigated the effect of TRH on polyphosphoinositide breakdown in frog pars intermedia. Neurointermediate lobes were labelled to isotopic equilibrium with myo-[3H]inositol.

TRH stimulated the rate of incorporation of [3H]inositol into the phospholipid fraction. The effect of TRH was concentration-dependent; half-maximal stimulation of α-MSH release and inositol incorporation occurred at 12 and 28 nmol TRH/1 respectively. In prelabelled neurointermediate lobes, lithium (10 mmol/l) enhanced the radioactivity in inositol monophosphate, bisphosphate (IP2) and trisphosphate (IP3). LiCl (10 mmol/l) induced a 38% inhibition of α-MSH release from perifused neurointermediate lobes but did not impair TRH-induced α-MSH secretion. In the presence of LiCl, TRH (1 μmol/l) induced a transient increase of the radioactivity in IP3, which was evident by 30 s and maximal by 1 min (+ 100%). TRH treatment also increased the radioactivity in IP2, which reached a plateau after 5 min (+ 100%). The increase in radioactivity in IP3 induced by TRH was closely paralleled by a rapid loss of [3H]phosphatidylinositol bisphosphate (PIP2), which was maximal by 1 min (−70%).

These results indicate that, in frog pars intermedia, TRH-evoked α-MSH secretion is coupled to breakdown of PIP2. The data suggest that, in amphibian melanotrophs, as previously shown in GH3 tumour cells and in rat pituitary mammotrophs, TRH causes rapid stimulation of polyphosphoinositide-hydrolysing phospholipase C.
The Booroola F gene mutation in sheep is not located close to the FSH-β gene
Authors: Montgomery G. W. , Sise J. A. , Greenwood P. J. , Fleming J. S.
Published: December 2008

A cDNA probe for the β subunit of bovine FSH (FSH-β) detects multiple restriction fragment length polymorphisms (RFLPs) in sheep genomic DNA consistent with an insertion/deletion polymorphism around the FSH-β locus. The presence of the insertion/deletion was confirmed by screening over 100 individuals with two restriction enzymes detecting RFLPs. All individuals showed the same patterns of fragments with both enzymes. A partial restriction map of the FSH-β gene in sheep suggests that the insertion/deletion is approximately 2 kb in size and located downstream from the third exon. Individual DNA samples were analysed from two flocks where the Booroola F gene is known to be segregating. Individuals that were heterozygous for the F gene were shown to be homozygous for one or other of the two alleles. Genetic recombination between the FSH-β locus and the F gene was observed in four pedigrees and there was no evidence that the insertion/deletion is closely linked genetically to the Booroola F gene. A major gene transcript of 2·2–2·3 kb was detected on Northern blots of sheep RNA. Neither the insertion/deletion polymorphism nor the presence of the F gene appeared to influence the size of the FSH-β gene transcript.
Glucocorticoids regulate the amount of G proteins in rat aorta
Authors: Haigh R. M. , Jones C. T. , Milligan G.
Published: December 2008

Glucocorticoids are known to influence cardiovascular sensitivity to catecholamines but the molecular mechanisms are undefined. We recently showed that glucocorticoids control the coupling of adrenergic receptors to G protein. Alterations in the amount of G protein is one mechanism by which receptor-G protein coupling may be controlled. Therefore, we set out to measure the levels of G proteins in aorta from normal, adrenalectomized and dexamethasonetreated adrenalectomized rats. G proteins were measured in plasma membrane preparations by immunoblotting and horseradish peroxidase staining. After adrenalectomy there was a 53% (n = 5) decrease in the density of staining for Gi (ANOVA; P<0.05 compared to controls). Conversely, there was a 210% (n = 5) increase in the density of staining for Gs. The levels of Go and the β-subunit of G proteins were not changed by adrenalectomy. Dexamethasone-replacement treatment after adrenalectomy returned Gi and Gs close to control values. Go remained unaltered compared to controls but was 24% (n=3) less than the adrenalectomized values (ANOVA; P<0.05). The levels of β-subunit after dexamethasone replacement were significantly greater (ANOVA; P<0.05) than both the controls and adrenalectomized values. These results show that glucocorticoids can differentially regulate the amounts of G proteins in rat aorta as in other tissues. This may be an important mechanism by which steroids control receptor-G protein coupling and hence transmembrane signalling pathways in vascular smooth muscle.
Internalization of human sex steroid-binding protein in the monkey epididymis
Authors: Gerard A. , Egloff M. , Gerard H. , Harate A. El , Domingo M. , Gueant J. L. , Dang C. D. , Degrelle H.
Published: December 2008

Human sex steroid-binding protein (hSBP) has been purified from late-pregnancy serum and labelled either by iodination (125I) or by photoaffinity with [3H]Δ6-testosterone. Using a micromanipulator, each labelled protein was separately injected into the lumen of epididymal tubules isolated from the head epididymis of the cynomolgus monkey (Macaca fascicularis). Tubules were sampled from 3 to 90 min after the injection and processed for electron microscope autoradiography. Localization of the label occurred over the epididymal epithelium whichever tracer was used. The labelling was not randomly distributed over the different cell types constituting the epithelium, since only the 'principal cells' exhibited a silver grain count significantly greater than the background count. In these cells, labelled protein was found over endocytic organelles (coated structures, endosomes, multivesicular bodies and the trans Golgi network) and nuclei (including the nuclear envelope). Quantitative analysis demonstrated the same pattern of cellular and subcellular distribution for each tracer. Pretreatment with excess unlabelled protein significantly reduced the uptake of radioactivity by the principal cells, demonstrating the specificity of this phenomenon. This is the first study to show direct histological evidence for the internalization of hSBP in the primate epididymis, consistent with earlier immunohistochemical or biochemical localization of this protein. It is concluded that head epididymal cells are able to take up labelled hSBP across their apical membrane. The mechanism of internalization seems to involve endocytosis by the principal cells and leads to labelling of the nuclear compartment. This is strikingly similar to the pattern of uptake of rat androgen-binding protein (rABP) by rat epididymal cells previously demonstrated by our group. To what extent the chemical and structural homology between hSBP and rABP can be held responsible for the common cytophysiological behaviour of these sex steroid-binding proteins remains to be determined.
Hypothalamo-pituitary regulation of the c-myc gene in rat liver
Authors: Hällstöm I. Porsch , Gustafsson J.-Å. , Blanck A.
Published: December 2008

Expression of the c-myc gene was studied in the livers of male and female Wistar rats. Furthermore, the effects on hepatic c-myc expression of neonatal and adult castration, with or without testosterone supplementation, as well as of continuous administration of GH to intact males, were analysed. Expression of c-myc was low in 6-day-old animals of both sexes, reached a maximum at 35 days of age and declined to the level of adult animals at 70 days. In prepubertal animals, expression was higher in females, but was higher in males after the onset of puberty, the postpubertal female rat liver exhibiting 50–70% of the expression in males.

Treatment of adult male rats with bovine GH in osmotic minipumps for 1 week reduced c-myc expression to the level of female rats. Castration, both neonatally and of adults, also feminized hepatic c-myc expression. Testosterone supplementation of the castrated animals increased the expression towards the level in sham-operated controls.

These results indicate that the c-myc gene is regulated by the hypothalamo-pituitary-liver axis via the sex-differentiated pattern of GH secretion, in analogy with other sex-differentiated hepatic functions, such as metabolism of steroids and xenobiotics. Neuroendocrine regulation of a gene such as c-myc, which is involved in the control of cell proliferation and differentiation, represents another aspect of the complex influence of GH on various somatic functions.