bluuebunny/crossref_metadata_2025 huggingface dataset:
Androgen receptors are only present in mesenchyme-derived dermal papilla cells of red deer (Cervus elaphus) neck follicles when raised androgens induce a mane in the breeding season
Authors: Thornton MJ , Hibberts NA , Street T , Brinklow BR , Loudon AS , Randall VA
Published: December 2004

Red deer stags produce an androgen-dependent mane of long hairs only in the breeding season; in the non-breeding season, when circulating androgen levels are low, the neck hair resembles the rest of the coat. This study was designed to determine whether androgen receptors are present in deer follicles throughout the year or only in the mane (neck) follicles when circulating testosterone levels are high in the breeding season. Although androgens regulate much human hair growth the mechanisms are not well understood; they are believed to act on the hair follicle epithelium via the mesenchyme-derived dermal papilla. The location of androgen receptors in the follicle was investigated by immunohistochemistry and androgen binding was measured biochemically in cultured dermal papilla cells derived from mane and flank follicles during the breeding season and from neck follicles during the non-breeding season. Immunohistochemistry of frozen skin sections using a polyclonal antibody to the androgen receptor localised nuclear staining only in the dermal papilla cells of mane follicles. Saturation analysis assays of 14 primary dermal papilla cell lines using [(3)H]-mibolerone demonstrated high-affinity, low-capacity androgen receptors were present only in mane (breeding season neck) cells; competition studies with other steroids confirmed the specificity of the receptors. Androgen receptors were not detectable in cells from either the breeding season flank nor the non-breeding season neck follicles. The unusual biological model offered by red deer of androgen-dependent hair being produced on the neck in the breeding, but not the non-breeding season, has allowed confirmation that androgen receptors are required in follicle dermal papilla cells for an androgen response; this concurs with previous human studies. In addition, the absence of receptors in the non-breeding season follicles demonstrates that receptors are not expressed unless the follicle is responding to androgens. Androgen receptors may be induced in mane follicles by seasonal changes in circulating hormone(s).
Differentiation of human parathyroid cells in culture
Authors: Liu W , Ridefelt P , Akerstrom G , Hellman P
Published: December 2004

Continuous culture of parathyroid cells has proven difficult, regardless from which species the cells are derived. In the present study, we have used a defined serum-free low calcium containing medium to culture human parathyroid cells obtained from patients with parathyroid adenomas due to primary hyperparathyroidism. No fibroblast overgrowth occurred, and the human parathyroid chief cells proliferated until confluent. After the first passage the cells ceased to proliferate, but still retained their functional capacity up to 60 days, demonstrated by Ca(2+)-sensitive changes in the release of parathyroid hormone (PTH) and as adequate cytoplasmic calcium ([Ca2+](i)) responses to changes in ambient calcium as measured by microfluorimetry. Low calcium concentrations enhanced, and vitamin D(3) and retinoic acids (RA) dose-dependently inhibited cell proliferation during the first passage, as determined by [(3)H]thymidine incorporation, immunohistochemistry for proliferating cell nuclear antigen and cell counting. Signs of differentiation were present as the set-points, defined as the external calcium concentration at which half-maximal stimulation of [Ca2+](i) (set-point(c)), or half-maximal inhibition of PTH release (set-point(p)) occur, were higher in not proliferating compared with proliferating cells in P0. Inhibition of cell proliferation was accompanied by signs of left-shifted set-points, indicating a link between proliferation and differentiation. The results demonstrate that human parathyroid chief cells cultured in a defined serum-free medium can be kept viable for a considerable time, and that signs of differentiation occur after proliferation has ceased. The low calcium stimulated cell proliferation may also be inhibited by vitamin D and RA.
The biological relevance of thyroid hormone receptors in immortalized human umbilical vein endothelial cells
Authors: Diekman MJ , Zandieh Doulabi B , Platvoet-Ter Schiphorst M , Fliers E , Bakker O , Wiersinga WM
Published: December 2004

The gene expression of thyroid hormone receptors (TR) in ECRF24 immortalized human umbilical vein endothelial cells (HUVECs) was investigated at both the mRNA and the protein level. Endothelin-1 (ET-1) and von Willebrand factor (vWF) production were measured in response to triiodothyronine (T(3)) administration. A real-time PCR technique was used to quantify the presence of mRNAs encoding for the different isoforms of the TR. The binding of T(3) to nuclear TRs was studied in isolated endothelial cell nuclei by Scatchard analysis. Expression of TR at the protein level was investigated by immunocytochemistry and Western blotting using TR-isoform-specific polyclonal rabbit antisera. ET-1 and vWF were measured in cell supernatants with a two-site immunoenzymatic assay. Scatchard analysis yielded a maximum binding capacity of 55 fmol T(3)/mg DNA (+/-200 sites/cell) with a K(d) of 125 pmol/l. Messenger RNAs encoding for the TRalpha1 and the TRalpha2 and the TRbeta1 were observed. The approximate number of mRNA molecules per cell was at least 50 molecules per cell for TRalpha1, five for TRalpha2 and two for TRbeta1. Immunocytochemistry revealed (peri)nuclear staining for TRbeta1, TRalpha1 and TRalpha2. ET-1 and vWF secretion did not increase upon addition of T(3) (10(-10)-10(-6) M). Immortalized ECRF24 HUVECs express TR, but at low levels. The number of TRs per endothelial cell is probably too low to be functional and no change in ET-1 or vWF production was found after addition of T(3). Therefore we conclude that the genomic effects of T(3) are unlikely to occur in these immortalized HUVECs.
Water metabolism disturbances at different stages of primary thyroid failure
Authors: Sahun M , Villabona C , Rosel P , Navarro MA , Ramon JM , Gomez JM , Soler J
Published: December 2004

The aim of the present study was to study salt and water metabolism in thyroid deficiency. We performed an oral water loading test (OWL) and a hypertonic 5% saline infusion test (HSI) in 16 patients with overt primary hypothyroidism before replacement treatment (PRE group) and after, in eight patients with subclinical hypothyroidism (SUB group) and in 16 normal individuals (CG group). In the PRE group, a lower free water clearance was detected in the OWL (P < 0.022), with lower plasma osmolality (OWL: P < 0.005; HSI: P < 0.001) and arginine vasopressin (AVP) (OWL: P < 0.001; HSI: P < 0.001) than the CG group, across both tests; they normalized with the replacement treatment. The same plasma abnormalities were detected in the SUB group with the HSI. Although the AVP and thirst thresholds did not differ between the groups, the lag between them was lower in the PRE (4.1+/-3.2 mOsm/kg) and SUB group (2.6+/-2.1 mOsm/kg) than in the CG group (13.3+/-9.2 mOsm/kg) (P < 0.05). There were no differences in atrial natriuretic hormone (ANH), plasma renin activity (PRA) and plasma aldosterone among the groups. These results indicate that plasma hypo-osmolality and low levels of AVP are present in primary hypothyroidism, and indeed are already present in the subclinical phase of the disease. An overlap between the thresholds of thirst and AVP seem to play a role in these abnormalities, but ANH, PRA and plasma aldosterone do not appear to contribute.
The role of the pituitary gland and ACTH in the regulation of mRNAs encoding proteins essential for adrenal steroidogenesis in the late-gestation ovine fetus
Authors: Simmonds PJ , Phillips ID , Poore KR , Coghill ID , Young IR , Canny BJ
Published: December 2004

To further understand the relative roles of the pituitary gland and ACTH in the regulation of mRNAs encoding proteins that are essential for adrenal development, we investigated the effects of, first, an ACTH infusion and labour in intact fetuses and, secondly, the effect of an ACTH infusion to fetuses with and without a pituitary gland, on the relative abundance of the mRNA encoding for the ACTH receptor (MC2R), steroidogenic factor 1 (SF-1), cholesterol side-chain cleavage enzyme (P450(scc)), 3beta-hydroxysteroid dehydrogenase (3betaHSD) and 17alpha-hydroxylase (P450(C17)) in the fetal adrenal gland. ACTH(1-24) infusion (14.7 pmol/kg per h) to intact fetuses was without effect on the abundance of mRNA encoding MC2R and SF-1, irrespective of whether the infusion was given for 18 (115-132 days of gestation) or 32 days (115 days to term (147 days of gestation)). Hypophysectomy (HX) did not alter the expression of MC2R mRNA; however, the abundance of SF-1 mRNA fell by approximately 50% following the removal of the pituitary gland. ACTH(1-24) infusion to HX fetuses failed to restore levels of SF-1 mRNA to that seen in intact animals. P450(scc) and 3betaHSD mRNAs were increased by ACTH(1-24) infusion for 18 days in intact animals, although no effects of the infusion were seen on P450(C17) mRNA levels. For all three of these mRNAs, there was a significant increase in their abundance between 132 days of gestation and term in intact fetuses. By term, ACTH(1-24) infusion was without any additional effect on their abundance. HX decreased the expression of P450(scc), 3betaHSD and P450(C17) mRNAs, while ACTH(1-24) infusion to HX fetuses increased the expression of these mRNAs to levels seen in intact animals. There were significant correlations between the abundance of the mRNA for P450(scc), 3betaHSD and P450(C17), but not MC2R and SF-1, and premortem plasma cortisol concentrations. These results emphasise the importance of the pituitary gland and ACTH in the regulation of the enzymes involved in adrenal steroidogenesis. Factors in addition to ACTH may also play some role, as the infusion was not always effective in increasing the abundance of the mRNAs. Surprisingly, the mRNA for MC2R and SF-1 did not appear to be regulated by ACTH in the late-gestation ovine fetus, though a pituitary-dependent factor may be involved in the regulation of SF-1 mRNA abundance.
Developmental regulation of corticotrophin receptor gene expression in the adrenal gland of the ovine fetus and newborn lamb: effects of hypoxia during late pregnancy
Authors: Fraser M , Braems GA ,   Challis
Published: December 2004

Responsiveness of the fetal sheep adrenal gland to adrenocorticotrophin (ACTH) increases in late pregnancy, resulting in increased glucocorticoid production. Development of this responsiveness is an important determinant of fetal hypothalamic-pituitary-adrenal function and depends, in part, on the potential for ACTH binding to adrenal tissue. In the present study, we have examined the developmental pattern of ACTH receptor (ACTH-R) expression during the latter half of pregnancy and in neonatal and adult life. As hypoxaemia induces increases in cortisol and ACTH secretion, in addition to increasing fetal adrenal responsiveness, a further aim of this study was to investigate whether hypoxaemia was associated with altered expression of the ACTH-R gene. Whole adrenal glands were removed from fetal sheep, lambs and adult sheep at different stages of development for measurement of ACTH-R mRNA. Moderate hypoxaemia was induced for 48 h beginning on days 124-128, or on days 132-134 of gestation, by decreasing the maternal fractional inspired oxygen. ACTH-R mRNA was detected by northern blotting using a cDNA cloned in our laboratory and by in situ hybridisation. ACTH-R mRNA (3.6 kb major transcript) was detected in adrenal tissue at day 63 of gestation. Its relative abundance increased significantly (P<0.05) between days 126-128 and 140-141 of pregnancy, increased further with the onset of spontaneous labour, and remained increased in newborn lambs at 7 h-7 days after birth. ACTH-R mRNA levels then decreased in adrenal tissue from lambs and adult sheep (P<0.05). Hypoxaemia for 48 h significantly increased ACTH-R mRNA expression in adrenals of the older fetuses (days 134-136) compared with that in controls (P<0.05), but was without effect in younger fetuses. We conclude that levels of ACTH-R mRNA in the fetal adrenal gland increase as term approaches, coincident with the endogenous prepartum surge in plasma ACTH and cortisol. Sustained hypoxaemia resulted in an upregulation of mRNA encoding for ACTH-R, but only in older fetuses and in association with a sustained increase in plasma cortisol. These results are consistent with cortisol, ACTH, or both, contributing to increased fetal adrenal responsiveness, by increasing expression of fetal adrenal receptors for ACTH.
Effects of sodium butyrate on expression of members of the IGF-binding protein superfamily in human mammary epithelial cells
Authors: Tsubaki J , Choi WK , Ingermann AR , Twigg SM , Kim HS , Rosenfeld RG , Oh Y
Published: December 2004

Dietary factors play an important role in both the development and prevention of human cancers, including breast carcinoma. One dietary micronutrient, sodium butyrate (NaB), is a major end product of dietary starch and fiber, produced naturally during digestion by anaerobic bacteria in the cecum and colon. NaB is a potent growth inhibitor and initiates cell differentiation for many cell types in vitro. In this study, we investigated the effects of NaB on three human mammary epithelial cells and regulation of the IGF axis, specifically, IGF-binding protein-3 (IGFBP-3), a known growth regulator in human mammary cells, and IGFBP-related protein 2 (IGFBP-rP2)/connective tissue growth factor. NaB inhibited DNA synthesis, as measured by [3H]thymidine incorporation, in estrogen-responsive (MCF-7) and estrogen-non-responsive (Hs578T) breast cancer cells, and normal human mammary epithelial cells (HMEC) to a similar degree (up to 90% inhibition at 1-10 mM concentrations). Treatment of cells with NaB induced histone hyperacetylation, suggesting that NaB exerts its biological effects, at least in part, as a histone deacetylase inhibitor in mammary epithelial cells. Treatment of Hs578T cells with NaB caused an induction of apoptotic cell death. NaB treatment resulted in increased levels of p21(Waf1/Cip1) mRNA and protein in Hs578T cells and distinct upregulation of p27(Kip1) in HMEC, suggesting that NaB activates different genes involved in cell cycle arrest, depending upon the cell type. In the same context, among the IGFBP superfamily members tested, NaB specifically upregulated the expression of IGFBP-3 and IGFBP-rP2. These two proteins are known to be involved in inhibition of mammary epithelial cell replication. Northern blot analysis showed that NaB treatment at 1-10 mM concentrations caused a dose-dependent stimulation of IGFBP-3 mRNA expression in cancerous cells and IGFBP-rP2 mRNA expression in both cancerous and non-cancerous cells. Protein data from Western ligand blot and immunoblot analyses demonstrated parallel results. In summary, we have demonstrated that NaB (i) uniformly suppresses DNA synthesis in both cancerous and non-cancerous mammary cells, and (ii) upregulates IGFBP-3 and IGFBP-rP2 mRNA and protein levels in cancerous and non-cancerous mammary cells. These results provide the first demonstration that butyrate regulates the IGFBP system in the human mammary system.
Localization of the IGF binding domain and evaluation of the role of cysteine residues in IGF binding in IGF binding protein-4
Authors: Byun D , Mohan S , Baylink DJ , Qin X
Published: December 2004

Our previous findings suggest that binding of IGF binding protein-4 (IGFBP-4) to IGFs is essential for the inhibitory effect of IGFBP-4 on the activity of IGFs, both in vitro and in vivo. Therefore, understanding the structural determinants of IGF binding in IGFBP-4 is important to the general understanding of the biology of the IGF system. This study sought to further localize the IGF binding domain and to evaluate the role of Cys residues in IGF binding. Our data revealed that full-length IGFBP-4 peptides lacking the residues Leu(72)-Ser(91) or Leu(72)-His(74) or Gly(75)-Ser(91) failed to bind to IGF-I or IGF-II, whereas deletion of the residue Leu(72) or residues Met(80)-Ser(91) led to a 2- to 3-fold reduction in IGF-I and IGF-II binding activity. The IGF-I and IGF-II binding activities were dramatically reduced by the single mutation, Cys9/Arg (>25-fold), and to a lesser degree, by the single mutation, Cys12/Arg (the first N-terminal Cys residue was designated Cys1). The mutation Cys17/Ser or Cys18/Tyr or Cys20/Ser each resulted in a similar but moderate ( approximately 5-fold) reduction in IGF-II binding activity. The IGF-I binding activity was also dramatically reduced by the mutation Cys18/Tyr, and to a lesser extent, by the mutation Cys17/Ser or Cys20/Ser. These data suggest: 1) the IGF-I and IGF-II binding domain in IGFBP-4 involves a hydrophobic motif (Leu(72)-Met(80)) located in the distal part of the conserved N-terminal region, and 2) the N-terminal Cys residues (Cys9 and Cys12) are more critical than the C-terminal Cys residues (Cys17 and Cys20) in affecting the IGF-I and IGF-II binding. Based on these data, we speculate that the structural determinants of IGF-I and IGF-II binding in IGFBP-4 are very similar, if not identical.
Beneficial effect of 1,25 dihydroxyvitamin D3 on cytokine-treated human pancreatic islets
Authors: Riachy R , Vandewalle B , Belaich S , Kerr-Conte J , Gmyr V , Zerimech F , d'Herbomez M , Lefebvre J , Pattou F
Published: December 2004

We examined whether 1,25 dihydroxyvitamin D(3) (1,25 D(3)), the active form of vitamin D involved in the regulation of the immune system, may also protect human pancreatic islet cells from destruction induced by cytokines. In this study, we specifically investigated the effect of 1,25 D(3) on oxidative stress and major histocompatibility complex (MHC) induction, both implicated in cytokine-induced islet cell dysfunction and destruction. We also investigated the effects of 1,25 D(3) on interleukin (IL)-6, a pleiotropic cytokine implicated in the pathogenesis of immunoinflammatory disorders. Human pancreatic islets, isolated from heart-beating donors, were treated with a combination of three cytokines, IL-1beta+tumor necrosis factor alpha+interferon gamma, in the presence or absence of vitamin D, and compared with with untreated control cells. Metabolic activity was assessed by cell viability and insulin content. Oxidative stress was estimated by heat shock protein 70 (hsp70) expression, cell manganese superoxide dismutase (MnSOD) activity and nitrite release, a reflexion of nitric oxide (NO) synthesis. Variation of immunogenicity of islet preparations was determined by analysis of the MHC class I and class II transcripts. Inflammatory status was evaluated by IL-6 production. After 48 h of contact with cytokines, insulin content was significantly decreased by 40% but cell viability was not altered. MHC expression significantly increased six- to sevenfold as well as NO and IL-6 release (two- to threefold enhancement). MnSOD activity was not significantly induced and hsp70 expression was not affected by the combination of cytokines. The addition of 1,25 D(3) significantly reduced nitrite release, IL-6 production and MHC class I expression which then became not significantly different from controls. These results suggest that the effect of 1,25 D(3) in human pancreatic islets cells may be a reduction of the vulnerability of cells to cytotoxic T lymphocytes and a reduction of cytotoxic challenge. Hence, 1,25 D(3) might play a role in the prevention of type 1 diabetes and islet allograft rejection.
Immunocytochemical demonstration of oestrogen receptor beta in blood vessels of the female rat
Authors: Andersson C , Lydrup ML , Ferno M , Idvall I , Gustafsson J , Nilsson BO
Published: December 2004

The role of oestrogen receptor (ER) beta in vascular function remains unclear. With the use of a specific ERbeta antibody we have now, using immunocytochemistry, visualized ERbeta in different parts of the vascular tree. In about 70% of medial smooth muscle cells of female rat aorta, tail artery and uterine artery, nuclear immunoreactivity to ERbeta was observed. In these vessels endothelial cells also expressed ERbeta. Vascular expression of the ERalpha subtype was lower than that of ERbeta. In aorta and tail artery, no immunoreactivity towards ERalpha was observed, while in uterine vessels occasional medial smooth muscle and endothelial cells expressed this ER subtype. ERbeta and alpha expression in uterine vessels was independent of the stage of the oestrous cycle, suggesting that variations in uterine blood flow occurring during the cycle are independent of ER density. The regional distribution of ERalpha, as determined by immunocytochemistry, was supported by measurements of ERalpha levels by enzyme immunoassay. In the uterine artery, the level of ERalpha was several times higher (P<0.001) than that of aorta and tail artery (10.1+/-1.7 fmol/mg protein in the uterine artery vs 3.3+/-1.0 and 0.5+/-0.5 fmol/mg protein in aorta and tail artery respectively). Thus, a prominent nuclear expression of ERbeta was observed in the vascular wall of several parts of the vascular tree, while ERalpha predominantly was expressed in uterine vessels, suggesting that ERbeta and alpha may have different roles in vascular function.
Testosterone receptor blockade restores cellular immunity in male mice after burn injury
Authors: Messingham KA , Messingham KA , Shirazi M , Duffner LA , Duffner LA , Emanuele MA , Kovacs EJ , Kovacs EJ , Kovacs EJ , Kovacs EJ
Published: December 2004

Males are known to have increased risk for septic complications after traumatic injury, which appears to be mediated by the inhibitory effects of testosterone on immune function. The role of testosterone in immunity after burn injury, however, remains unclear. Herein, we examined the effects of a testosterone receptor antagonist, flutamide, on delayed type hypersensitivity response (DTH), splenocyte proliferation, interleukin (IL)-2 secretion, and IL-2 receptor (IL-2R) expression in male BALB/c mice subjected to a 15% total body surface area burn or sham injury. Burn- or sham-injured mice were given flutamide s.c. at 30 min and 24 h after injury. At 48 h, burn injury caused a 48% (P<0.001) decrease in DTH response; however, mice that received flutamide treatment did not demonstrate significant suppression of DTH. Likewise, splenocyte proliferation and IL-2 production were depressed in burned animals in comparison with sham-injured controls, and flutamide treatment resulted in a partial restoration of these responses. In vitro studies indicated that splenocytes from sham- and burn-injured mice were equally sensitive to the suppressive effects of 5alpha-dihydrotestosterone in regard to proliferation and IL-2 production. Further evaluation revealed a decrease in IL-2R expression on splenocytes from burned mice and a partial restoration of this expression with flutamide treatment. Thus blocking testosterone receptor activation improves the cellular immunity in thermally injured mice, possibly through restoration of IL-2 production and IL-2R expression. It remains to be determined whether the effects of testosterone in this injury model are direct or indirect.
The steroid receptor antagonists RU40555 and RU486 activate glucocorticoid receptor translocation and are not excreted by the steroid hormones transporter in L929 cells
Authors: Pariante CM , Pearce BD , Pisell TL , Su C , Miller AH
Published: December 2004

RU40555 is a recently available glucocorticoid receptor (GR) antagonist that differs from RU486 by a methyl radical. We have used the mouse fibroblast cell line L929 to study the in vitro effects of RU40555 on GR translocation and function and on the membrane steroid hormones transporter. The results showed that: 1) RU40555 competed for the binding of labelled dexamethasone (Dex) with a K(i) of 2.4 nM; 2) both RU40555 and RU486 were equally potent inhibitors of Dex-induced GR-mediated gene transcription; 3) maximum GR translocation induced by micromolar concentrations of Dex and the GR antagonists was approximately 30-55% loss in the cytoplasmic GR and approximately 40-90% increase in the nuclear GR (assessed by GR immunostaining in cytoplasm and nucleus and western blots of immunoprecipitated GR protein in cytosolic and nuclear fractions) and was similar for the two antagonists; 4) at nanomolar concentrations, RU40555 and RU486 induced more GR translocation than Dex (assessed by [(3)H]Dex binding and western blot of immunoreactive GR in the same cytosolic homogenates); 5) blocking the steroids membrane transporter with verapamil (100 microM) in the presence of Dex (10 nM) increased GR translocation to levels similar to those induced by RU40555 (10 nM) and RU486 (10 nM) alone; 6) verapamil did not affect GR translocation in the presence of RU40555 or RU486. These data demonstrate similar quantitative effects on GR translocation by RU486 and the new GR antagonist, RU40555. Moreover, RU40555, like RU486, is an effective GR antagonist. Finally, there is no evidence that the intracellular concentrations of RU40555 or RU486 are regulated by the steroids membrane transporter in L929 cells.
Extracellular matrix regulates ovine granulosa cell survival, proliferation and steroidogenesis: relationships between cell shape and function
Authors: Huet C , Pisselet C , Mandon-Pepin B , Monget P , Monniaux D
Published: December 2004

The extracellular matrix (ECM), constituting the follicular basal lamina and present also between follicular cells and in the follicular fluid, is believed to regulate granulosa cell (GC) function during follicular development. Ovine GCs isolated from small (1-3 mm in diameter) or large (4-7 mm in diameter) antral follicles were cultured on various pure ECM components (type I collagen, fibronectin, laminin), synthetic substrata enhancing (RGD peptides) or impairing (poly 2-hydroxyethylmethacrylate (poly-hema)) cell adhesion, or in the presence of heparin. The effects of these factors, used alone or in combination with IGF-I and/or FSH, were evaluated in terms of GC spread, survival, proliferation and steroidogenesis. When grown on type I collagen (CI) gel, poly-hema or heparin, GCs from both large and small follicles exhibited a round shape and a low proliferation rate. Compared with non-coated plastic substratum as a control, these ECM or synthetic compounds enhanced estradiol secretion and reduced progesterone secretion by large-follicle GCs. In contrast, GCs from both large and small follicles spread extensively on CI coating, fibronectin, laminin and RGD peptides. Fibronectin and laminin dramatically increased the proliferation rate and enhanced survival of GCs from both origins. Moreover, fibronectin, laminin and RGD peptides reduced estradiol secretion by large-follicle GCs. Unexpectedly, CI coating increased estradiol secretion and reduced progesterone secretion by large-follicle GCs, suggesting that type I collagen was able to maintain estradiol secretion independently of GC shape. Finally, GC responsiveness to IGF-I and FSH, in terms of proliferation and steroidogenesis, was generally maintained when cells were grown on ECM components, RGD peptides and in the presence of heparin. However, when large-follicle GCs were grown as non-adherent clusters (as observed on poly-hema) basal and IGF-I- and/or FSH-stimulated progesterone secretions were totally abolished. Overall, this study shows that GC shape, survival, proliferation and steroidogenesis can be modulated in vitro by pure ECM components in a specific and coordinated manner. It is suggested that, in vivo, fibronectin and laminin would sustain follicular development by enhancing the survival and proliferation of GCs, whereas type I collagen might participate in the maintenance of estradiol secretion in large antral follicles.
Myocardial gene expression of glucose transporter 1 and glucose transporter 4 in response to uteroplacental insufficiency in the rat
Authors: Tsirka AE , Gruetzmacher EM , Kelley DE , Ritov VH , Devaskar SU , Lane RH
Published: December 2004

Uteroplacental insufficiency causes intrauterine growth retardation (IUGR) and subsequent low birth weight, which predisposes the affected newborn towards adult Syndrome X. Individuals with Syndrome X suffer increased morbidity from adult ischemic heart disease. Myocardial ischemia initiates a defensive increase in cardiac glucose metabolism, and individuals with Syndrome X demonstrate reduced insulin sensitivity and reduced glucose uptake. Glucose transporters GLUT1 and GLUT4 facilitate glucose uptake across cardiac plasma membranes, and hexokinase II (HKII) is the predominant hexokinase isoform in adult cardiac tissue. We therefore hypothesized that GLUT1, GLUT4 and HKII gene expression would be reduced in heart muscle of growth-retarded rats, and that reduced gene expression would result in reduced myocardial glucose uptake. To prove this hypothesis, we measured cardiac GLUT1 and GLUT4 mRNA and protein in control IUGR rat hearts at day 21 and at day 120 of life. HKII mRNA quantification and 2-deoxyglucose-uptake studies were performed in day-120 control and IUGR cardiac muscle. Both GLUT1 and GLUT4 mRNA and protein were significantly reduced at day 21 and at day 120 of life in IUGR hearts. HKII mRNA was also reduced at day 120. Similarly, both basal and insulin-stimulated glucose uptake were significantly reduced in day-120 IUGR cardiac muscle. We conclude that adult rats showing IUGR as a result of uteroplacental insufficiency express significantly less cardiac GLUT1 and GLUT4 mRNA and protein than control animals (which underwent sham operations), and that this decrease in gene expression occurs in parallel with reduced myocardial glucose uptake. We speculate that this reduced GLUT gene expression and glucose uptake contribute towards mortality from ischemic heart disease seen in adults born with IUGR.
Effects of adenosine 5'-triphosphate and growth hormone on cellular H+ transport and calcium ion concentrations in cloned bovine mammary epithelial cells
Authors: Katoh K , Komatsu T , Yonekura S , Ishiwata H , Hagino A , Obara Y
Published: December 2004

The present experiment was carried out to investigate the effects of exogenous adenosine 5'-triphosphate (ATP) and growth hormone (GH) on cellular H(+) efflux rate (extracellular acidification rate) and Ca(2+) concentration ([Ca(2+)](c)) in cloned bovine mammary epithelial cells (bMEC) raised from the mammary gland of a 26-day-pregnant Holstein heifer. Perifusion of 2-day cultured cells with a medium containing ATP (10, 100 and 1000 micromol/l) for 30 min caused a significant and concentration-dependent increase in the cellular H(+) efflux rate. ATP application (100 micromol/l) caused a transient and large increase in [Ca(2+)](c) in all cells. In contrast, perifusion with a medium containing bovine GH at 10, 50 and 250 ng/ml for 30 min caused a significant decrease in the cellular H(+) efflux rate in a concentration-dependent manner. However, bovine GH application (50 ng/ml) caused a small decrease followed by an increase, in some cases, in [Ca(2+)](c). In bMEC treated with lactogenic hormones (1 microgram/l prolactin, 1 nmol/ml dexamethasone and 5 microgram/ml insulin) for 2 days, the increased H(+) efflux rate induced by ATP was significantly reduced, whereas the negative response induced by GH was inversely and significantly changed to the positive. Treatment of the cells with lactogenic hormones reduced the increase in [Ca(2+)](c) induced by ATP stimulation, while it enhanced the increase in [Ca(2+)](c) induced by GH stimulation. Application of ATP or GH did not cause any significant changes in [pH](c). Treatment with lactogenic hormones enhanced GH receptor (GHR) transcription that was determined by RT-PCR. From these results, we conclude that exogenous application of ATP and GH causes prompt and significant responses in H(+) transport and [Ca(2+)](c) that were significantly changed in the opposite direction by the treatment with lactogenic hormones. The lactogenic hormone treatment also enhanced GHR transcription, which may change post-receptor signal transduction systems for both agents in the bMEC.
Shedding of growth hormone-binding protein is inhibited by hydroxamic acid-based protease inhibitors: proposed mechanism of activation of growth hormone-binding protein secretase
Authors: Amit T , Amit T , Hochberg Z , Yogev-Falach M , Youdim MB , Youdim MB , Barkey RJ
Published: December 2004

The present study describes events postulated to be involved in the regulated mechanism of proteolytic shedding of growth hormone (GH)-binding protein (GHBP). Using Chinese hamster ovary (CHO) cell lines stably transfected either with the full-length human GH receptor (hGHR) or with the cytoplasmic domain-truncated hGHR (hGHR(tr)), we show that the phorbol ester, phorbol 12-myristate 13-acetate (PMA), caused a rapid time- and dose-dependent increase in GHBP secretion, which, as expected, was matched by a corresponding decrease in cell-surface GHR. Furthermore, PMA equally enhanced GHBP release from CHO/hGHR(tr) cells, suggesting that the cytoplasmic domain of hGHR is not essential for PMA-induced shedding. PMA is known to specifically activate protein kinase C and, indeed, the stimulatory effects of PMA in both cell lines were completely inhibited by the protein kinase inhibitor, staurosporine (100 nM), suggesting that activation of protein kinase C (PKC) may mediate PMA-induced GHBP shedding. Since proteolytic cleavage of several cell-surface proteins was shown to be stimulated by modulators of PKC activity and inhibited by metalloprotease inhibitors, we studied the effects of two hydroxamic acid-based inhibitors of zinc-dependent metalloproteases, BB-3103 and Ro31-9790, on GHBP proteolysis. Pretreatment of CHO/hGHR cells with both these inhibitors reduced PMA-enhanced shedding of GHBP, in a dose-dependent manner, with IC(50) values of approximately 0.41 microM for BB-3103 and approximately 0.97 microM for Ro31-9790. In addition, these inhibitors dose-dependently reduced the shedding enhanced by the sulfhydryl alkylator, N-ethylmaleimide (NEM), with IC(50) values of approximately 0.32 microM and approximately 0.58 microM for BB-3103 and Ro31-9790 respectively. It was of interest to find out that Ro31-9790 acted not only to modulate PMA- or NEM-induced shedding processes, but also markedly reduced the spontaneous, time-dependent accumulation of GHBP released from CHO/hGHR cells growing in serum-containing medium. Taken together, these results suggest that one or more zinc-dependent metalloprotease(s), acting at the cell surface, may be involved in GHBP secretase activity. A scheme is proposed whereby at least part of the regulated maturation and/or activation of the protease activity may involve a cysteine-switch mechanism and/or PKC-dependent phosphorylation. In the long run, specific inhibitors of these processes could be applied in the regulation of GHBP levels and, thus, of GH availability and/or activity.
Seasonal fluctuations in pituitary levels of the three forms of gonadotropin-releasing hormone in striped bass, Morone saxatilis (Teleostei), during juvenile and pubertal development
Authors: Holland MC , Hassin S , Zohar Y
Published: December 2004

In most vertebrates, the development of a mature gonadotropin-releasing hormone (GnRH) secretory system is pivotal for the onset of puberty. The role of the three native GnRH forms, seabream (sb) GnRH, chicken (c) GnRH-II and salmon GnRH, in striped bass puberty remains elusive. This study examined the changes in pituitary GnRH levels throughout juvenile and pubertal development, a period encompassing 3 to 4 years. The levels of the two most abundant forms in the pituitary, sbGnRH and cGnRH-II (10:1), increased during the Fall and peaked prior to (cGnRH-II) or during (sbGnRH) the natural breeding season in March to May. In most cases, sbGnRH and cGnRH-II levels of maturing fish correlated to changes in oocyte diameter, gonadosomatic index and LH pituitary content. Interestingly, pituitaries of immature and maturing 2- and 3-year-old males and females contained similar amounts of all three GnRH forms. Additionally, pituitary sbGnRH and cGnRH-II levels in juvenile fish were relatively high and GnRH profiles showed a clear seasonality, similar to those of older, mature fish. These findings suggest a role for both sbGnRH and cGnRH-II in the regulation of gonadal development and indicate that, unlike some mammalian species, the timing of puberty in striped bass is not limited by a low activity of the GnRH system.
Aldosterone secretion by the rat adrenal cortex is stimulated by the activation of protease-activated receptor 1
Authors: Raven PW , Kapas S , Carroll M , Hinson JP
Published: December 2004

Stimulation of aldosterone by a serine protease, trypsin, was first reported in 1982, although the mechanism of this effect was unclear. Recently, a family of protease-activated receptors (PARs) has been described and four members of the family characterised and cloned, including the previously recognised thrombin receptor. This study investigated whether PARs mediate the action of trypsin on aldosterone secretion. Using intact rat adrenal capsular tissue, thrombin was found to increase aldosterone secretion, and the effects of trypsin on aldosterone secretion were confirmed. Both trypsin and thrombin were shown to activate phospholipase C, as measured by an increase in inositol triphosphate turnover by adrenal capsular tissue. It was also shown that U73122, a phospholipase C inhibitor, attenuated the aldosterone response to trypsin. These effects were consistent with the activation of a PAR. Northern blot analysis revealed the presence of mRNA encoding PAR-1, but not PARs-2, -3 or -4 in the adrenal capsule/zona glomerulosa. Messenger RNA encoding PAR-1 was increased by dietary sodium depletion, consistent with previous reports of an increased response to trypsin after sodium depletion. These data suggest that the actions of trypsin on aldosterone secretion are mediated by PAR-1.
Subcellular distribution and glycosylation pattern of androgen receptor from sheep omental adipose tissue
Authors: McCann JP , Mayes JS , Hendricks GR , Harjo JB , Watson GH
Published: December 2004

Sex steroids are known to have an influence on the distribution, metabolism and accretion of adipose tissue. These steroids carry out their function via specific receptors. We have previously reported the presence of oestrogen and progesterone receptors in sheep adipose tissues. In this study, we have tested the subcellular distribution of androgen receptor (AR) in sheep omental adipose tissue. Subcellular fractions - microsomes, plasma membrane and nuclei-cell debris - were isolated by differential and sucrose gradient centrifugation and confirmed by electron microscopy. The AR was determined in each fraction by Western blot analyses. As anticipated, the receptor was found in the cytosolic fraction, but a high concentration was also present in the microsomal fraction, a lesser amount in the plasma membrane fraction, and only a small amount was left in the nuclei-cell debris fraction. Two minor immunostaining bands with approximate molecular weights of 250 and 140 kDa and a major band at 110 kDa were detected in the cytosolic fraction, but only the 110 kDa band was detected in the membrane fractions. A 104 kDa band was observed on occasion and believed to be a degradation product. The cytosolic isoforms were tested for sensitivity to glycosidases. This treatment resulted in a decrease in the amount of the 250 and 140 kDa bands. To substantiate that the 250 and 140 kDa isoforms were glycoproteins, the cytosolic fraction was chromatographed on Concanavalin A-Sepharose. The 110 kDa band was eluted in the 0.4 M KCl salt wash while the 250 and 140 kDa bands were eluted with alpha-methylmannoside. Treatment of the glycoprotein (alpha-methylmannoside) peak with glycosidases converted the 250 and 140 kDa bands to the 110 kDa band. These data confirm the presence of AR in subcellular fractions of adipose tissue and suggest that it exists in various glycosylated isoforms.
TSH-induced differentiated functions correlate with enhancement of phosphotyrosine phosphatase activity in thyroid cells. Effect of phorbol 12-myristate 13-acetate
Authors: Petitfrere E , Huet E , Sartelet H , Martiny L , Legue O , Haye B
Published: December 2004

TSH-treated pig thyroid cells reorganize into follicle-like structures and exhibit differentiated functions. TSH also induces a phosphotyrosine phosphatase (PTPase) activity evaluated by phosphorylated substrate hydrolysis. Incubation of thyrocytes with various concentrations of 8-bromo-cyclic AMP or forskolin induces an increase of PTPase activity in a dose-dependent manner. During the culture period, adenylyl cyclase sensitivity, protein binding iodine and PTPase activity progressively increase from the first to the fourth day of the culture. Chronic treatment with phorbol 12-myristate 13-acetate (PMA) significantly inhibits PTPase activity during the first 24 h following PMA addition. GF 109203X, a specific inhibitor of protein kinase C, abolishes the inhibitory effect of PMA. Electrophoresis of membrane extracts allowed us to demonstrate a phosphatase activity at 111 kDa (p111). Vanadate inhibits this activity, indicating that p111 is a PTPase. This p111 is significantly reduced in PMA-treated cells. These data suggest that PTPase activity evidenced at 111 kDa is correlated with a differentiated state of primary cultured pig thyroid cells induced by TSH.
The Booroola (FecB) phenotype is associated with a mutation in the bone morphogenetic receptor type 1 B (BMPR1B) gene
Authors: Souza CJ , MacDougall C , MacDougall C , Campbell BK , McNeilly AS , Baird DT
Published: December 2004

Genetic variations in ovulation rate which occur in different breeds of sheep provide useful models to explore the mechanisms regulating the development of antral follicles. The Booroola gene, an autosomal mutation that affects ovulation rate, has been known for over two decades and despite intensive research it has not yet been identified. Using resources from human genome mapping and known data about gene linkage and chromosome location in the sheep, we selected the gene encoding the Bone Morphogenetic Protein receptor (BMPR) type 1 B (ALK-6) as a candidate site for the mutation. The BMPR1B gene in the human is located at the region linked with the Booroola mutation, syntenic to chromosome 6 in the sheep. A fragment of the sheep BMPR1B gene was cloned from an ovarian cDNA and the deduced aminoacid (AA) sequence is over 98% homologous to the known mammalian sequences. cDNA and genomic DNA from 20 Booroola genotypes were screened and two point mutation were found in the kinase domain of the receptor, one at base 746 of the coding region (A in the ++ to a G in FF animals) which results in a change from a glutamine in the wild type to a arginine in the Booroola animals. Another point mutation was identified at position 1113, (C to A) but this mutation does not change the coding aminoacid. The first mutation was confirmed in genomic DNA from 10 ewes from an independent Brazilian flock which segregates the Booroola phenotype. In all instances homozygous FecB gene carrier (n=11) had only the 746 A to G mutation, non gene carriers (n=14) had only the wild type sequence and heterozygote gene carriers (n=5) had both sequences. This mutation in the subdomain 3 of the kinase domain could result in an alteration in the expression and/or phosphorylation of SMADs, resulting in the phenotype characteristic of the Booroola animals which is the 'precocious' development of a large number of small antral follicles resulting in increased ovulation rate.
Insulin, growth hormone and sport
Authors: Sonksen PH
Published: December 2004

This review examines some interesting 'new' histories of insulin and reviews our current understanding of its physiological actions and synergy with GH in the regulation of metabolism and body composition. It reviews the history of GH abuse that antedates by many years the awareness of endocrinologists to its potent anabolic actions. Promising methods for detection of GH abuse have been developed but have yet to be sufficiently well validated to be ready for introduction into competitive sport. So far, there are two promising avenues for detecting GH abuse. The first uses immunoassays that can distinguish the isomers of pituitary-derived GH from the monomer of recombinant human GH. The second works through demonstrating circulating concentrations of one or more GH-sensitive substances that exceed the extremes of normal physiological variability. Both methods require blood rather than urine samples. The first method has a window of opportunity lasting about 24 h after an injection and is most suitable for 'out of competition' testing. The second method has reasonable sensitivity for as long as 2 weeks after the last injection of GH and is uninfluenced by extreme exercise and suitable for post-competition samples. This method has a greater sensitivity in men than in women. The specificity of both methods seems acceptably high but lawyers need to decide what level of scientific probability is needed to obtain a conviction. Both methods need further validation before implementation. Research work carried out as part of the fight against doping in sport has opened up a new and exciting area of endocrinology.
Differential regulation of the TRH gene promoter by triiodothyronine and dexamethasone in pancreatic islets
Authors: Fragner P , Lee SL , Aratan de Leon S
Published: December 2004

TRH was initially found in the hypothalamus and regulates TSH secretion. TRH is also produced by insulin-containing beta-cells. Endogenous TRH positively regulates glucagon secretion and attenuates pancreatic exocrine secretion. We have previously shown that triiodothyronine (T(3)) down-regulates pre-pro-TRH gene expression in vivo and in vitro. The present study was designed to determine the initial impact of T(3) on rat TRH gene promoter and to compare this effect with that of dexamethasone (Dex). Primary islet cells and neoplastic cells (HIT T-15 and RIN m5F) were transiently transfected with fragments of the 5'-flanking sequence of TRH fused to the luciferase reporter gene. The persistence of high TRH concentrations in fetal islets in culture, probably due to transactivating factors, allowed us to explore how T(3) and Dex regulate the TRH promoter activity in transfected cells and whether the hormone effect is dependent on the cell type considered. TRH gene promoter activity is inhibited by T(3) in primary but not neoplastic cells and stimulated by Dex in both primary and neoplastic cells of islets. These findings validate previous in vivo and in vitro studies and indicate the transcriptional impact of these hormones on TRH gene expression in the pancreatic islets.