bluuebunny/crossref_metadata_2025 huggingface dataset:
Exogenous expression of Pit-1 in AtT-20 corticotropic cells induces endogenous growth hormone gene transcription
Authors: Kurotani R , Yoshimura S , Iwasaki Y , Inoue K , Teramoto A , Osamura RY
Published: December 2004

The pituitary-specific POU-homeodomain transcription factor, Pit-1, is known to regulate the expression of the GH gene in somatotropes, prolactin (PRL) in lactotropes, and TSH in thyrotropes. It is not normally expressed in corticotropes or gonadotropes. We addressed the question of whether exogenous Pit-1 was sufficient to induce ectopic transcription of the GH gene in the corticotropic cell line, AtT-20, or the gonadotropic cell line, alpha T3-1. A fusion gene composed of enhanced green fluorescent protein gene and human Pit-1 cDNA was transfected into AtT-20 and alpha T3-1 cells. The endogenous mouse GH mRNA was induced in three of nine AtT-20 cell lines and one of three alpha T3-1 cell lines containing the fusion gene. A small amount of GH protein was also detected in these cell lines. These data indicate that transfected Pit-1 is capable of inducing transcription of the GH gene in AtT-20 cells and alpha T3-1 cells. These data also suggest that synergistic co-factors might be required to transcribe the GH gene effectively for translation into GH protein. Furthermore, our findings support the hypothesis that the function of anterior pituitary cells is determined by the combinatorial action of specific transcription factors.
Analysis of expression of chorionic gonadotrophin transcripts in prostate cancer by quantitative Taqman and a modified molecular beacon RT-PCR
Authors: Span PN , Thomas CM , Heuvel JJ , Bosch RR , Schalken JA , vd Locht L , Mensink EJ , Sweep CG
Published: December 2004

Expression of human chorionic gonadotrophin (hCG) is associated with trophoblastic, testicular and other malignancies such as bladder, pancreatic, cervical, breast and prostate cancer. In the prostate, however, hCG expression, associated with neuroendocrine cells, is also found in normal tissue. Of the six highly homologous genes that all encode the beta-subunit of hCG, the beta 7 gene is reportedly the only gene expressed in several non-transformed tissues. The beta 3, 5 and 8 genes would be variably expressed in malignant tissue and placenta, but not in normal tissue. To assess to what extent this expression difference can also be found in the prostate, we compared the levels of the different hCG beta transcripts in concurrent normal and cancerous prostate tissues obtained from 17 patients. To this end, we developed a Taqman real-time fluorescent RT-PCR assay for hCG beta, and a quantitative assay specific for the beta 3, 5 and 8 genes, modified from the molecular beacon principle. This latter assay proved highly specific and capable of reliably distinguishing between these hCG beta transcripts that differ in only one nucleotide. Surprisingly, median expression levels of hCG beta were lower in prostate cancer when compared with normal tissue from the same patient. In contrast, hCG beta 3, 5 and 8 transcripts were found in normal tissue and did not differ in prostate cancer, arguing against a specific role of these transcripts in the development of prostate cancer.
Myometrial expression of 11 beta-hydroxysteroid dehydrogenase type 2 in rat pregnancy
Authors: Burton PJ , Waddell BJ
Published: December 2004

The enzyme 11 beta-hydroxysteroid dehydrogenase type 2 (11 beta-HSD2), which reduces glucocorticoid potency in target cells by metabolism of active glucocorticoids, is expressed in the non-pregnant rat uterus in an oestrogen-dependent manner. Because glucocorticoids appear to facilitate parturition in many species, expression of 11 beta-HSD2 in pregnant myometrium is likely to influence pregnancy maintenance and possibly the onset and progression of labour. The present study therefore examined myometrial 11 beta-HSD2 mRNA, protein and bioactivity across rat pregnancy, with emphasis on the peripartum period. A single 1.9 kb transcript of 11 beta-HSD2 mRNA was evident in myometrium at all stages, with maximal (P<0.05) levels observed at day 16 (term=day 23). Consistent with this pattern of mRNA expression, Western blot analysis showed the presence of a 40 kDa 11 beta-HSD2 protein at all stages, with the maximal immunoreactive signal also observed on day 16. The 11 beta-HSD2 signal was immunolocalized to myometrial smooth muscle cells and endometrial stromal cells. Bioactivity specific to 11 beta-HSD2 was detectable in myometrium at all stages, but in contrast to the patterns of 11 beta-HSD2 mRNA and protein, the V(max) decreased at the beginning of pregnancy and remained stable until term. The apparent K(m) of 11 beta-HSD2 for corticosterone increased from 47 +/- 11 nM in non-pregnant myometrium to 75 +/- 7 nM by day 10 of pregnancy, and remained high until returning to an intermediate level on the day of delivery (60 +/- 8 nM). Progesterone competitively inhibited 11 beta-HSD2 bioactivity (K(i)=1.75 muM) whereas 20 alpha-hydroxypregn-4-en-3-one, the other major progestin present during rat pregnancy, had no such effect. In conclusion, these data suggest that local levels of active glucocorticoid in the myometrium are determined by the net effects of myometrial 11 beta-HSD-1 and -2 expression across pregnancy. Because the previously reported increase in myometrial 11 beta-HSD-1 near term occurs with little change in myometrial 11 beta-HSD2 bioactivity, this is likely to facilitate parturition by increasing local concentrations of active glucocorticoid.
Pachytene spermatocytes in co-culture inhibit rat Sertoli cell synthesis of inhibin beta B-subunit and inhibin B but not the inhibin alpha-subunit
Authors: Clifton RJ , O'Donnell L , Robertson DM
Published: December 2004

This study investigates the effects of spermatogenic germ cells on inhibin alpha-subunit and beta B-subunit expression, and inhibin alpha-subunit and inhibin B production by rat Sertoli cells in vitro. Sertoli cells isolated from 19-day-old rats were cultured for 48 h at 32 degrees C, in the presence or absence of FSH (2.3-2350 mIU/ml), and in the presence of pachytene spermatocytes, round spermatids or cytoplasts of elongated spermatids purified from adult rat testis by elutriation and density gradient separation. Sertoli cell secretion of inhibin alpha-subunit and inhibin B, as measured by immunoassay, was dose-dependently stimulated by FSH (maximal stimulation 13- and 2-fold, respectively). Round spermatids or cytoplasts co-cultured with Sertoli cells had no effect on basal or FSH-induced secretion of inhibin alpha-subunit or inhibin B. When Sertoli cells were co-cultured with pachytene spermatocytes, inhibin alpha-subunit secretion was unaltered, while inhibin B secretion was suppressed in a cell concentration-dependent manner to reach a maximal suppression of 45% compared with Sertoli cells alone (P<0.01). A similar suppression in inhibin B was still observed (64% of Sertoli cells alone) when the pachytene spermatocytes were separated from Sertoli cells by a 0.45 microm pore membrane barrier in bicameral chambers. Pachytene spermatocytes also suppressed FSH-induced inhibin B levels in Sertoli cell co-cultures and this suppression was attributed to a decrease in basal inhibin B production rather than a change in FSH responsiveness. Quantitation of Sertoli cell inhibin alpha- and beta B-subunit mRNA by quantitative (real-time) PCR demonstrated that pachytene spermatocytes did not alter Sertoli cell alpha-subunit mRNA expression, but significantly (P<0.01) suppressed basal and FSH-induced beta B-subunit mRNA expression to a similar degree to that seen with inhibin B protein levels. It is concluded that pachytene spermatocytes in vitro suppress Sertoli cell inhibin B secretion via factor-mediated suppression of inhibin beta B-subunit expression. These findings support the hypothesis that specific germ cell types can influence inhibin B secretion by the testis independent of FSH regulation.
Overexpression of vasopressin in the rat transgenic for the metallothionein-vasopressin fusion gene
Authors: Nagasaki H , Yokoi H , Arima H , Hirabayashi M , Ishizaki S , Tachikawa K , Murase T , Miura Y , Oiso Y
Published: December 2004

Arginine vasopressin (AVP) is a major antidiuretic hormone, the overproduction of which causes diluting hyponatremia in humans and is called the syndrome of inappropriate antidiuresis (SIAD). To study physiological changes resulting from AVP overproduction and to develop an animal model of hyponatremia, the human AVP gene was expressed under the control of the metallothionein promoter in transgenic (Tg) rats. Analyses of AVP immunoreactivity (irAVP) in the tissues revealed that the transgene is expressed mainly in the central nervous system. Gel filtration showed that irAVP in the brain and plasma was properly processed AVP. AVP purified from the brains of both Tg and control rats also exerted equal bioactivity to generate cAMP in LLC-PK1 cells. The founder rats did not show any physical or anatomical abnormalities. Under basal conditions, Tg rats had high plasma AVP levels (Tg 13.8 +/- 2.5 pg/ml; control 2.7 +/- 1.2 pg/ml; n=6 in both groups; means +/- S.E.M.), decreased urine volume, and normal plasma [Na(+)]. Hypertonic saline injected i.p. did not affect AVP secretion in Tg rats. In response to a zinc-supplemented liquid diet, plasma AVP decreased in control rats, but increased in Tg rats (Tg 32.7 +/- 2.7 pg/ml; control 1.0+/-0.1 pg/ml; n=6), resulting in hyponatremia (Tg 135.2 +/- 2.5 mEq/l; control 140.8 +/- 0.4 mEq/l; n=6). To our knowledge, this is the first transgenic animal to show diluting hyponatremia. This transgenic rat may therefore provide a useful model in which to investigate various physiological alterations resulting from the oversecretion of AVP which involve SIAD, stress response, behavior, and blood pressure.
Adrenopause or decline of serum adrenal androgens with age in women living at sea level or at high altitude
Authors: Gonzales GF , Gonez C , Villena A
Published: December 2004

The present study aimed to determine adrenopause or reduction of serum adrenal androgens with age at high altitude and at sea level. It was a cross-sectional study performed in 210 women resident at high altitude (4340 m) and 123 women living in Lima (150 m), aged 20-70 years. Fasting early morning blood samples were obtained. Serum dehydroepiandrosterone (DHEA), DHEA sulphate (DHEAS), androstenedione, testosterone and estradiol were measured by radioimmunoassay. Serum testosterone concentrations were greater in women living at high altitude than in those resident at sea level. Serum concentrations of DHEA, DHEAS and androstenedione were lower in women living at high altitude than in those living at sea level. The DHEAS/DHEA ratio was significantly greater, and the androstenedione/testosterone ratio was lower in samples from women living at high altitude. Among women older than 50 years of age, a greater decline in serum concentrations of DHEA was observed in those living at high altitude than in those living at sea level. Among women 60-70 years of age, serum concentrations of DHEA at high altitude were 46.9% of those in women of the same age living at sea level. Decay of DHEAS at sea level and at high altitude occurred from the age of 40 years. The decline was faster at high altitude than at sea level, and in women aged 60-70 years serum values of DHEAS at high altitude were 56% of those at sea level. In the same age group, serum concentrations of androstenedione among those native to high altitudes were 27.34% of the value at sea level. At sea level, serum testosterone concentrations did not change with age from 20 to 70 years. In women aged 20-39 years and 50-59 years, serum testosterone concentrations were greater at high altitude than at sea level (P<0.05). In those aged 60-70 years, the concentrations were similar in those living at sea level and at high altitude. At sea level and at high altitude, the serum testosterone/estradiol ratio increased with age (P<0.0034 and P<0.0001 respectively). This ratio increased at an earlier age among those living at high altitude (40-49 years) than among those living at sea level (50-59 years). Multivariate analysis showed that altitude (P<0.0001) and greater chronological age (P<0.001) were associated with lower serum DHEAS concentrations. DHEAS was related to chronological age (P<0.0001). Low serum androstenedione concentrations were related to living at high altitude at birth and greater chronological age (P<0.0001). In conclusion, adrenopause is attained earlier and is of greater magnitude at high altitude than at sea level.
Effects of thyroid hormones on pulmonary and renal angiotensin-converting enzyme concentrations in fetal sheep near term
Authors: Forhead AJ , Fowden AL
Published: December 2004

In the sheep fetus, pulmonary and renal concentrations of angiotensin-converting enzyme (ACE) increase towards term in parallel with the prepartum surges in plasma cortisol and tri-iodothyronine (T(3)). The ontogenic change in pulmonary ACE has been shown to be induced, at least in part, by cortisol but the role of the thyroid hormones is unknown. Therefore, this study investigated the effects of thyroid hormones on tissue ACE concentration in fetal sheep during late gestation. Pulmonary and renal ACE concentrations were measured in sheep fetuses after experimental manipulation of thyroid hormone status by fetal thyroidectomy and exogenous hormone infusion. In intact fetuses, pulmonary and renal ACE concentrations increased between 127-132 and 142-145 days of gestation (term 145 +/- 2 days), coincident with the prepartum rises in plasma cortisol and T(3). The ontogenic increment in pulmonary ACE concentration was abolished when the prepartum surge in T(3), but not cortisol, was prevented by fetal thyroidectomy. At 143-145 days, ACE concentration in the lungs and kidneys of the thyroidectomised fetuses were both lower than those in the intact fetuses. In intact fetuses at 127-132 days, pulmonary ACE was upregulated by intravenous infusions of either cortisol (2-3 mg/kg per day) or T(3) (8-12 microg/kg per day) for 5 days. Renal ACE was unaffected by cortisol or T(3) infusion. Therefore, thyroid hormones have an important role in the developmental control of pulmonary and renal ACE concentration in the sheep fetus towards term. In addition, the prepartum rise in plasma T(3) appears to mediate, in part, the maturational effect of cortisol on pulmonary ACE concentration.
Effects of dibutyryl cAMP on stanniocalcin and stanniocalcin-related protein mRNA expression in neuroblastoma cells
Authors: Wong CK , Yeung HY , Mak NK , DiMattia GE , Chan DK , Wagner GF
Published: December 2004

Stanniocalcin is a polypeptide hormone that was first reported in fish as a regulator of mineral metabolism. Its recent identification in mammals has opened a new area of investigation in basic and clinical endocrinology. In the present study, regulation of the stanniocalcin (STC) and stanniocalcin related protein (STCrP) genes were investigated in mouse neuroblastoma cells (Neuro-2A) in relation to neuronal cell differentiation. Neuro-2A is an undifferentiated cell line that contains measurable levels of STCrP mRNA, but undetectable levels of STC mRNA. Treatment of the cells with either dbcAMP (1-4 mM) or 50 microM euxanthone (PW1) resulted in extensive differentiation and neurite outgrowth. However, only neurites of dbcAMP-treated cells developed varicosities, a phenotypic marker of axon formation. Furthermore, following differentiation induced by dbcAMP, there was an upregulation of STC and downregulation of STCrP mRNA levels. In the first 24 and 48 h of treatments, there was a maximum twofold induction and 1.5-fold reduction in STC and STCrP mRNAs respectively. Following 96 h of treatment, an additional 14-fold STC induction and 1.2-fold STCrP reduction were observed. The increase in STC mRNA levels was accompanied by a concomitant increase in axon-specific low molecular form microtubule-associated protein (MAP-2c) mRNA and varicosities on the neurites, suggesting a possible role for STC in axonogenesis. There was no induction of STC mRNA levels when PW1 was added into the culture media, whereas ionomycin (1-10 microM) had no observable effects on cell differentiation or STC/STCrP mRNA. Immunocytochemical staining of dbcAMP-treated cells revealed abundant levels of immunoreactive STC, particularly in the varicosities, with only weak staining in control, untreated cells. Antisense oligodeoxynucleotides transfection studies indicated that the expression of STC was a cause of varicosity formation and a consequence of cell differentiation. Our findings lend further support to the notion that STC is involved in the process of neural differentiation.
Cardiac contractile function is enhanced in isolated ventricular myocytes from growth hormone transgenic mice
Authors: Colligan PB , Brown-Borg HM , Duan J , Ren BH , Ren J
Published: December 2004

Growth hormone (GH) plays a key role in cardiac growth and function. However, excessive levels of GH often result in cardiac dysfunction, which is the major cause of death in acromegalic patients. Transgenic mice with GH over-expression serve as useful models for acromegaly and exhibit impaired cardiac functions using echocardiography, similar to those of human acromegaly. However, the mechanism underscoring the impaired ventricular function has not been well defined. This study was designed to evaluate the cardiac excitation-contraction coupling in GH over-expressing transgenic mice at the single ventricular myocyte level. Myocytes were isolated from GH and age-matched wild-type mouse hearts. Mechanical properties were evaluated using an IonOptix MyoCam system. The contractile properties analyzed included peak shortening (PS), time-to-peak shortening (TPS) and time-to-90% relengthening (TR(90)), and maximal velocities of shortening/relengthening (+/-dL/dt). Intracellular Ca2+ properties were evaluated by fura-2. GH transgenic mice exhibited significantly increased body weights and enlarged heart and myocyte size. Myocytes from GH transgenic mice displayed significantly enhanced PS and+/-dL/dt associated with similar TPS and TR(90) compared with the wild-type littermates. Myocytes from GH transgenic mice displayed a similar resting intracellular Ca2+ level and Ca2+ removal rate but exhibited an elevated peak intracellular Ca2+ level compared with the wild-type group. Myocytes from both groups were equally responsive to increases in extracellular Ca2+ concentration and stimulating frequency. These results suggest that GH over-expression is associated with enhanced contractile function in isolated myocytes and that the impaired cardiac function observed in whole hearts may not be due to defects at the myocyte level.
Adenovirus-mediated gene transfer in the ovine pituitary gland is associated with hypophysitis
Authors:   Davis, McMahon RF , Lowenstein PR , Castro MG , Lincoln GA , McNeilly AS
Published: December 2004

Gene therapy for pituitary disease requires evaluation for safety as well as efficacy. We have reported results of adenovirus-mediated gene transfer using the sheep as a large animal model that allows longitudinal evaluation of hormone secretion and have confirmed high levels of transgene expression up to 7 days after direct stereotaxic injection into the pituitary gland. Here we report the results of detailed histological examination of the pituitary glands from animals injected with two recombinant adenoviruses expressing the beta-galactosidase marker gene, or with saline vehicle to control for the potential tissue-disruptive effect of the injection volume itself. Pituitaries injected with saline showed no evidence of inflammatory response apart from occasional minor foci of apoptosis. In all other respects they were indistinguishable from normal uninjected control pituitary glands. Glands injected with recombinant adenoviruses containing either the hCMV-beta-gal or the hPRL-beta-gal transgene, on the other hand, displayed variable degrees of inflammatory response, with periglandular fibrosis, lymphocytic infiltrate and venulitis in almost all cases. Focal necrosis and/or apoptosis was noted in six of nine cases. In summary, we have found evidence of severe inflammatory reaction within the first seven days of adenovirus injection, amounting to significant hypophysitis. The histological extent of this reaction has not previously been recognised by studies of the efficacy of gene transfer in rodents, and was underestimated by immunocytochemical studies of hormone and transgene expression. The findings emphasise the need for careful evaluation of the safety of endocrine gene therapy, and for caution with the dose of vector used.
NFkappaB1 (p50)-deficient mice are not susceptible to multiple low-dose streptozotocin-induced diabetes
Authors: Mabley JG , Hasko G , Liaudet L , Soriano F , Southan GJ , Salzman AL , Szabo C
Published: December 2004

Insulin-dependent diabetes mellitus (IDDM) is a disease characterized by the autoimmune destruction of the pancreatic beta-cells, which requires the expression of a number of immune-related genes including major histocompatibility complex proteins, cytokines, chemokines, and cytotoxic enzymes, many of which are regulated by the transcription factor, NFkappaB. Inhibition of the entire NFkappaB family of transcription factors may be harmful, as these factors are involved in many normal physiological processes. However, identifying and targeting specific NFkappaB subunits critical for the pathogenesis of disease may prove to be valuable in designing new therapeutic strategies. To assess the potential role of the NFkappaB subunit, p50, in the development of IDDM, mice with gene disruption for NFkappaB (p50) were investigated for susceptibility to IDDM. We found that p50-deficient mice were fully resistant against multiple low-dose streptozotocin-induced diabetes, a model of diabetes with a strong autoimmune component. The site of involvement of NFkappaB (p50) lies at an early, critical juncture of immune activation and proinflammatory mediator production, because: (1) isolated islets of Langerhans from NFkappaB (p50)-deficient mice were not protected from the islet dysfunction induced by in vitro application of proinflammatory cytokines; (2) p50-deficient mice were not resistant to diabetes induced by a single high dose of streptozotocin, a model with a large oxidant component and no autoimmune involvement; and (3) diabetes induced up-regulation of nitric oxide and interleukin-12 was blocked in the p50-deficient mice. Our data suggest that NFkappaB (p50) has an essential role in the development of autoimmune diabetes. Selective therapeutic blockade of this subunit may be beneficial in preventing IDDM.
Calcium inhibits ovarian steroidogenesis in the blowfly Phormia regina
Authors: Maniere G , Vanhems E , Gautron F , Delbecque JP
Published: December 2004

Calcium is frequently involved in the stimulation of steroidogenesis in gonads and endocrine glands, generally in association with cAMP. However, our present observations show that it has the opposite effect in the ovary of the blowfly Phormia regina. Our in vitro experiments first showed that extracellular calcium does not play a role during the stimulation of steroidogenesis in fly ovaries; indeed steroidogenesis was activated in vitro as efficiently in a medium with or without calcium, either by pharmacological compounds mimicking cAMP signaling or by active brain extracts. When calcium was experimentally introduced into biosynthetic cells by ionophores or liberated from internal stores by thapsigargin, it did not activate, but clearly inhibited both basal and acute steroidogenesis respectively in previtellogenic and in vitellogenic ovaries. Our experiments also demonstrated that calcium decreases cAMP concentrations in the ovaries of Phormia, by stimulating its degradation, without modifying its biosynthesis. Moreover, inhibitors of calcium-calmodulin phosphodiesterases (PDEs) increased steroid biosynthesis in vitro, whereas inhibitors of calcium-insensitive PDEs did not. These data thus demonstrate that, in blowfly ovaries, calcium ions inhibit cAMP-stimulated steroidogenesis by activating a calmodulin-sensitive (type I) PDE.
Direct effects of ciliary neurotrophic factor on brown adipocytes: evidence for a role in peripheral regulation of energy homeostasis
Authors: Ott V , Fasshauer M , Dalski A , Klein HH , Klein J
Published: December 2004

Ciliary neurotrophic factor (CNTF) plays an important role in regulating neuronal growth. Recently, central anorexigenic effects of this cytokine have been characterized. However, peripheral effects on tissues that actively contribute to the regulation of energy homeostasis have not been described. Here, we report direct potent and selective effects of CNTF on growth factor and metabolic signalling intermediates in mouse brown adipocytes. CNTF stimulates STAT3, MAP kinase, Akt, and p70 S6 kinase. We find that, next to mediating Akt and p70 S6 kinase activation, both phosphatidylinositol 3-kinase and protein kinase C are separately acting, main intermediates for inducing mitogen-activated protein (MAP) kinase activation. On a functional level, CNTF enhances beta3-adrenergic induction of uncoupling protein-1. Thus, these results demonstrate direct effects of CNTF on adipose tissue signalling and metabolism and suggest a novel role for this cytokine in the peripheral regulation of energy homeostasis.
Statin-induced apoptosis of vascular endothelial cells is blocked by dexamethasone
Authors: Newton CJ , Ran G , Xie YX , Bilko D , Burgoyne CH , Adams I , Abidia A , McCollum PT , Atkin SL
Published: December 2004

Statins block de novo synthesis of cholesterol by inhibiting the enzyme, HMG CoA reductase. The product of this reaction, mevalonic acid, is also a precursor of isoprenoids, molecules required for the activation of signalling G-proteins, such as Ras. Signal transduction pathways involving Ras are important for cell survival and this may be why statins induce apoptotic death of several cell types. Given that statins are used to treat vascular disease, it is surprising that no studies have been conducted on vascular endothelial cells. For this reason, we have tested the effect of fluvastatin (FS) on the endothelial cell line EA.hy 926. Here we show that FS, at concentrations from 1 to 2 microM, blocks growth and induces apoptosis of the endothelial cell line, EA.hy 926. As considerable redundancy exists in cell signalling pathways for cell survival, toxicity of FS under more physiological conditions might be prevented by pathways that do not require Ras, such as those activated by adrenal or sex steroids. To test this hypothesis, first RT-PCR analysis was performed for nuclear receptor mRNA expression. This revealed the presence of mRNA for the androgen receptor (AR) and glucocorticoid receptor (GR). The effect of the AR agonist, dihydrotestosterone (DHT), and the GR agonist, dexamethasone (Dex), was then tested. Whilst DHT (100 nM) had no effect on FS-induced cell death, Dex (1 microM) blocked FS-induced apoptosis. Cell cycle analysis revealed that 24 h exposure to FS prevented cells from leaving G(1) and 24-48 h later a marked sub-G(1) peak was observed. Dex was able to reduce the sub-G(1) peak, but it failed to reduce accumulation of cells in G(1). Further studies revealed that, in addition to blocking FS-induced apoptosis, Dex was able to block apoptosis of EA.hy 926 cells induced by serum deprivation, tumour necrosis factor-alpha, oxidants, DNA damage and mitochondrial disruption. This study strongly suggests that glucocorticoids have a role to play in preventing vascular injury and they may provide a reason why statins are apparently not toxic to vascular endothelial cells in vivo.
Prolactin-releasing peptide in the ewe: cDNA cloning, mRNA distribution and effects on prolactin secretion in vitro and in vivo
Authors: Curlewis JD , Kusters DH , Barclay JL , Anderson ST
Published: December 2004

RT-PCR followed by 5'- and 3'- rapid amplification of cDNA ends was used to clone and sequence ovine prolactin-releasing peptide (PrRP). The cDNA was characterised by short 5'- and 3'-untranslated regions and a GC-rich (71%) coding region. The nucleotide and deduced amino acid sequences for the coding region showed 95.6 and 94.9% identity with bovine PrRP but the amino acid sequence of PrRP31 was conserved between these species. Northern blot analysis and RT-PCR showed that, as in the rat, the peptide was more abundantly expressed in the brainstem than the hypothalamus. However, in the ovine hypothalamus, PrRP mRNA expression was more widespread than in the rat, with expression detected in both rostral and caudal parts of the mediobasal hypothalamus. The effects of synthetic ovine PrRP on prolactin secretion both in vitro and in vivo were also examined. In primary cultures of sheep pituitary cells, PrRP significantly (P<0.01) increased prolactin concentrations in the culture medium but the response was not observed in every experiment and was only seen when pituitary glands were dispersed with collagenase rather than trypsin. PrRP was much less potent than TRH which caused a significant (P<0.01) two- to threefold increase in prolactin concentrations in every experiment. Intravenous (10 and 50 nmol) or intracerebroventricular (10 and 50 nmol) injection of PrRP had no significant effect on either plasma prolactin concentration or pulsatile LH secretion whereas intravenous injection of TRH (10 nmol) produced a highly significant (P<0.01) and more than sevenfold stimulation of plasma prolactin concentrations. In conclusion, these results suggest that PrRP is unlikely to be an important prolactin-releasing factor in this species.
Photoperiod-dependent regulation of inhibin in Siberian hamsters: II. Regulation of inhibin production and secretion by pregnant mare serum gonadotropin
Authors: Kenny HA , Bernard DJ , Horton TH , Woodruff TK
Published: December 2004

Inhibin production differs in ovaries of Siberian hamsters (Phodopus sungorus) exposed to long days (LD) or short days (SD). We believe that seasonal differences in serum follicle-stimulating hormone contribute to this difference. However, given the profound photoperiodic differences in follicle maturation, serum gonadotropins alone may not account for all of the observed differences in inhibin processing. To test this hypothesis, we challenged LD and SD female hamsters with exogenous gonadotropins. While both groups responded with increased inhibin expression, the effects were muted in ovaries of SD females and there was no evidence of ovulation in these animals. These data indicate that the ovaries of SD females are not immediately equipped to respond to gonadotropin stimulation. More generally, these data suggest that photoperiodic history affects ovarian inhibin production and secretion in response to gonadotropins.
Novel role for prostaglandin E2 in fish hepatocytes: regulation of glucose metabolism
Authors: Busby ER , Cooper GA , Mommsen TP
Published: December 2004

Prostaglandin E(2) (PGE(2)) potently activated glycogenolysis and gluconeogenesis in isolated rockfish (Sebastes caurinus) hepatocytes. The average degree of activation for glycogenolysis was 6.4+/-0.67-fold (mean+/-S.E.M.; n=37), and could be as much as 19-fold. Analysis of dose-concentration relationships between glycogenolytic actions and PGE(2) concentrations yielded an EC(50) around 120 nM in hepatocyte suspensions and 2 nM for hepatocytes immobilized on perifusion columns. For the activation of gluconeogenesis (1.74+/-0.14-fold; n=10), the EC(50) for suspensions was 60 nM. Intracellular targets for PGE(2) actions are adenylyl cyclase, protein kinase A and glycogen phosphorylase. Concentrations of cAMP increased with increasing concentrations of PGE(2), and peaked within 2 min of hormone application. In the presence of the phosphodiesterase inhibitor, isobutyl-3-methylxanthine, peak height was increased and peak duration extended. The protein kinase A inhibitor, Rp-cAMPS, counteracted the activation of glycogenolysis by PGE(2), implying that the adenylyl cyclase/protein kinase A pathway is the most important, if not exclusive, route of message transduction. PGE(2) activated plasma membrane adenylyl cyclase and hepatocyte glycogen phosphorylase in a dose-dependent manner. The effects were specific for PGE(2); smaller degrees of activation of glycogenolysis were noted for PGE(1), 11-deoxy PGE(1), 19-R-hydroxy-PGE(2), and prostaglandins of the A, B and Falpha-series. The selective EP(2)-receptor agonist, butaprost, was as effective as PGE(2), suggesting that rockfish liver contains prostaglandin receptors pharmacologically related to the EP(2) receptors of non-hepatic tissues of mammals. Rockfish hepatocytes quickly degraded added PGE(2) (t((1/2))=17-26 min). A similar ability to degrade PGE(2) has been noted in catfish (Ameiurus nebulosus) hepatocytes, but no glycogenolytic or gluconeogenic actions of the hormone are noted for this species. We conclude that PGE(2) is an important metabolic hormone in fish liver, with cAMP-mediated actions on glycogen and glucose metabolism, and probably other pathways regulated by cAMP and protein kinase A. The constant presence of EP(2)-like receptors is a unique feature of the fish liver, with interesting implications for function and evolution of prostaglandin receptors in vertebrates.
Regulation of rat Sertoli cell function by FSH: possible role of phosphatidylinositol 3-kinase/protein kinase B pathway
Authors: Meroni SB , Riera MF , Pellizzari EH , Cigorraga SB
Published: December 2004

The FSH molecular mechanism of action is best recognized for its stimulation of the adenylyl cyclase/cAMP pathway via activation of a G protein. Recently, links between cAMP, phosphatidylinositol 3-kinase (PI3K) and protein kinase B (PKB) signaling pathways in thyroid and granulosa cells have been observed. The aim of this study was to investigate the possible role of the PI3K/PKB pathway in FSH regulation of Sertoli cell function. Twenty-day-old rat Sertoli cell cultures were used. An increase in phosphorylated PKB (P-PKB) levels in response to FSH and dibutyryl-cAMP was observed. These increments in P-PKB levels were not observed in the presence of two PI3K inhibitors, wortmannin and Ly 294002. Inhibition of protein kinase A (PKA) by H89 did not decrease FSH stimulation of P-PKB levels. Taken together, these results indicate that FSH increases P-PKB levels in a PI3K-dependent and PKA-independent manner in rat Sertoli cells. In addition, wortmannin partially inhibited the ability of FSH to stimulate two well-known parameters of Sertoli cell function - transferrin secretion and lactate production - at doses equal to or lower than 0.1 microM. Related to lactate production, a decrease in FSH stimulation of lactate dehydrogenase activity and of basal and FSH-stimulated glucose uptake was observed in the presence of wortmannin. These metabolic changes were in most cases accompanied by changes in the levels of P-PKB. Altogether, these results suggest a meaningful role of the PI3K/PKB pathway in the mechanism of action of FSH in rat Sertoli cells.
The late gestation increase in circulating ACTH and cortisol in the fetal sheep is suppressed by intracerebroventricular infusion of recombinant ovine leptin
Authors: Howe DC , Gertler A ,   Challis
Published: December 2004

The obese gene product leptin, originally characterised as an adipocyte hormone coordinating the behavioural and neuroendocrine responses to starvation, is expressed in fetal adipocytes and placental trophoblast cells and is present in the fetal circulation. Concentrations of leptin in fetal blood correlate with fetal bodyweight and fat mass. In post-natal life, leptin conveys information about calorie intake and the state of adipose tissue energy stores, and plasma leptin levels are generally inversely correlated with hypothalamo-pituitary adrenal (HPA) activity. Late fetal life is characterised by increasing HPA activity that prepares the fetus for extrauterine life and initiates the endocrine cascade leading to parturition. We have investigated the hypothesis that leptin in the fetal circulation can inhibit the fetal HPA axis, thereby providing a mechanism by which the fetus can determine the fine timing of parturition as long as it is adequately nourished and growing appropriately. Here we show that a 5-day intracerebroventricular infusion of leptin to the sheep fetus in late gestation inhibits the pre-parturient rise in ACTH and cortisol concentrations, and that this seems to be a centrally mediated effect.
Insulin increases the density of potassium channels in white adipocytes: possible role in adipogenesis
Authors: Ramirez-Ponce MP , Mateos JC , Bellido JA
Published: December 2004

We studied the potassium currents in white adipocytes obtained by culturing preadipocytes from rat epididymal tissue, both with insulin (WA(i)) and without insulin (WA(o)), in order to test the role of insulin in the development of voltage-gated potassium channels (K(v)) during adipogenesis. Occasionally, very small potassium currents (I(K,V)) were present in preadipocytes; however these currents were measured in all differentiated cells (adipocytes). WA(i) exhibited greater macroscopic potassium currents than WA(o) with no apparent differences in kinetics or voltage dependence. The current density (pA/ micro m(2)) calculated in WA(i) was higher than in WA(o). Currents were blocked by millimolar concentrations of tetrethylamonium (TEA). The effect of insulin on adipogenesis, both with and without TEA, was analysed. Four days without insulin and three days with insulin were necessary to increase the total number of cells in culture by 2.5-fold. Insulin increased the number of differentiated cells by 73.5%. Cell proliferation and differentiation were inhibited by TEA. Proliferation was affected only by high concentration of TEA. Inhibition of differentiation was dose dependent, with the concentration necessary for half-block similar to the IC(50) values to block potassium channels. These results suggest that insulin increases the density of K(v) and that these channels may be necessary for the normal growth of white adipocytes in culture.
Corticotrophin-releasing hormone inhibits insulin-like growth factor-I release from primary cultures of rat granulosa cells
Authors: Calogero AE , Barreca A , Burrello N , Palermo I , Giordano G , D'Agata R , Vicari E
Published: December 2004

Corticotrophin-releasing hormone (CRH), a neuropeptide which modulates gonadal function during stress, is expressed by several cell types of the rat ovary and is able to suppress oestrogen release from rat granulosa cells. The mechanism of this effect is, however, not known. Since insulin-like growth factor (IGF)-I is produced by rat granulosa cells and exerts a synergistic role with FSH on granulosa cell steroidogenesis, we hypothesised that CRH may suppress oestrogen release from granulosa cells by inhibiting IGF-I release and/or stimulating the release of its binding protein (IGFBP-3). To test this hypothesis, granulosa cells were obtained from immature female Sprague-Dawley rats primed with diethylstilboestrol, and hormone concentrations were measured in the conditioned medium by radioimmunoassay. CRH suppressed oestrogen and IGF-I release stimulated by FSH used at a concentration of 1 IU/l, whereas it did not have any statistically significant effect on oestrogen and IGF-I release in basal conditions or in response to 5 IU/l FSH. The suppressive effects of CRH on oestrogen and IGF-I release were antagonised by a selective CRH receptor antagonist. CRH had no effects on IGFBP-3 release. CRH did not have any effect on oestrogen release stimulated by increasing concentrations of IGF-I and its suppressive effect on FSH-stimulated oestrogen release was overcome by the addition of low doses of exogenous IGF-I. In conclusion, CRH suppressed the release of oestrogen and IGF-I, but not of IGFBP-3. Thus, the inhibitory effects of CRH on oestrogen release could be mediated, partly, by a suppression of the autocrine/paracrine action of IGF-I.
The Interferon Stimulated Genes (ISG) 17 and Mx have different temporal and spatial expression in the ovine uterus suggesting more complex regulation of the Mx gene
Authors: Johnson GA , Joyce MM , Yankey SJ , Hansen TR , Ott TL
Published: December 2004

Interferon stimulated gene 17 (ISG17) and Mx are up-regulated in the ruminant uterus in response to interferon-tau (IFNtau) during early pregnancy. Recent evidence strongly indicates that expression of ISGs occur only in stroma (ST) and glandular epithelium (GE) during this time as a result of transcriptional repression by interferon regulatory factor two (IRF-2) expression in the LE. The present report tested this hypothesis by examining mRNA and protein expression of ISG17 and Mx in serial uterine cross-sections obtained from cyclic and early pregnant ewes. In situ and immunocytochemical analysis revealed that ISG17 mRNA and protein were low to undetectable, whereas Mx mRNA was expressed in the lumenal (LE) and superficial GE at all days of the estrous cycle examined. Both ISG17 and Mx mRNA increased in the stratum compactum ST between Days 11 and 13, and expression extended into the deep GE and stratum spongiosum ST on Days 15 through 17 in pregnant ewes. Interestingly the Mx gene continued to be strongly expressed in LE and superficial GE through Day 17 of pregnancy, whereas ISG17 remained low to undetectable in these cells. Collectively, this study highlights the complexity of the uterine environment by unequivocally illustrating differential temporal and spatial expression of the IFN-responsive genes ISG17 and Mx.
Liver receptor homologue-1 is expressed in human steroidogenic tissues and activates transcription of genes encoding steroidogenic enzymes
Authors: Sirianni R , Seely JB , Attia G , Stocco DM , Carr BR , Pezzi V , Rainey WE
Published: December 2004

In the current study we test the hypothesis that liver receptor homologue-1 (LRH; designated NR5A2) is involved in the regulation of steroid hormone production. The potential role of LRH was assessed by first examining expression in human steroidogenic tissues and second by examining effects on transcription of genes encoding enzymes involved in steroidogenesis. LRH is closely related to steroidogenic factor 1 (SF1; designated NR5A1), which is expressed in most steroidogenic tissues and regulates expression of several steroid-metabolizing enzymes. LRH transcripts were expressed at high levels in the human ovary and testis. Adrenal and placenta expressed much lower levels of LRH than either ovary or liver. To examine the effects of LRH on steroidogenic capacity we used reporter constructs prepared with the 5'-flanking region of steroidogenic acute regulatory protein (StAR), cholesterol side-chain cleavage (CYP11A1), 3beta hydroxysteroid dehydrogenase type II (HSD3B2), 17alpha hydroxylase, 17,20 lyase (CYP17), 11beta hydroxylase (CYP11B1) and aldosterone synthase (CYP11B2). Co-transfection of these reporter constructs with LRH expression vector demonstrated that like SF1, LRH enhanced reporter activity driven by flanking DNA from StAR, CYP11A1, CYP17, HSD3B2, and CYP11B1. Reporter constructs driven by CYP11A1 and CYP17 were increased the most by co-transfection with LRH and SF1. Of the promoters examined only HSD3B2 was more sensitive to LRH than SF1. The high level of ovarian and testicular LRH expression make it likely that LRH plays an important role in the regulation of gonadal function.
Specifying the cellular responses to IGF signals: roles of IGF-binding proteins
Authors: Duan C
Published: December 2004

Regulation of peptide growth factor/hormone activities by secreted hormone-binding proteins has emerged as a common theme in cell-cell signaling. Among the best-studied examples are members of the IGF-binding protein (IGFBP) gene family. These secreted proteins bind the IGF ligands with equal or even greater affinities than do the IGF receptors, and therefore are placed in a critical regulatory position between IGFs and their cell surface receptors. The circulating IGF/IGFBP complexes prolong the half-lives of IGFs and buffer the potential hypoglycemic effects of IGFs. Locally expressed IGFBPs provide a means of localizing IGFs in specific cells and can alter the IGF biological activity. While some members of the IGFBP gene family have been consistently shown to inhibit IGF actions by preventing them from gaining access to the IGF receptors, others potentiate IGF actions by facilitating the ligand-receptor interaction. Furthermore, recent studies indicate that some IGFBPs can regulate several cellular processes through ligand-independent mechanisms. This review will focus on the roles of IGFBPs in vascular smooth muscle cells. A conceptual model of the molecular mechanisms by which IGFBPs act to determine the specific physiological outcomes of IGF stimulation is proposed and discussed.
Bone defects in latent TGF-beta binding protein (Ltbp)-3 null mice; a role for Ltbp in TGF-beta presentation
Authors: Dabovic B , Chen Y , Colarossi C , Zambuto L , Obata H , Rifkin DB
Published: December 2004

The latent transforming growth factor (TGF)-beta binding proteins (LTBP)-1, -3 and -4 bind the latent form of the multipotent cytokine TGF-beta. To examine the function of the LTBPs, we made a null mutation of Ltbp-3 by gene targeting. The homozygous mutant animals developed cranio-facial malformations by 12 days. By three months, there was a pronounced rounding of the cranial vault, extension of the mandible beyond the maxilla, and kyphosis. The mutant animals developed osteosclerosis of the long bones and vertebrae as well as osteoarthritis between 6 and 9 months of age. These latter phenotypic changes were similar to those described for mice that have impaired TGF-beta signaling. Thus, we suggest that Ltbp-3 plays an important role in regulating TGF-beta bioavailability as the phenotype of the Ltbp-3 null mouse appears to result from decreased TGF-beta signaling. Histological examination of the skulls from null animals revealed no effects on calvarial suture closure. However, the synchondroses in the skull base were obliterated within 2 weeks of birth. This is in contrast to the wild-type synchondroses, which remain unossified throughout the life of the animal and enable growth of the skull base through endochondral ossification. Histological changes in mutant basooccipital-basosphenoid synchondrosis were observed 1.5 days after birth. Compared with wild-type or heterozygous littermates, the basooccipital-basosphenoid synchondrosis of Ltbp-3 null mice contained increased numbers of hypertrophic chondrocytes. The expression of bone sialoprotein-1 (a marker for osteoblasts) was observed in cells surrounding the synchondrosis at postnatal day 1.5 indicating ectopic ossification. The expression of Indian hedgehog (Ihh) (a marker for chondrocytes committed to hypertrophic differentiation) was found through the basooccipital-basosphenoid synchondrosis, whereas the expression of parathyroid hormone related protein (PTHrP), which inhibits chondrocyte differentiation, appeared to be diminished in Ltbp-3 null mice. This suggests that Ltbp-3 may control chondrocyte differentiation by regulating TGF-beta availability. TGF-beta may regulate PTHrP expression either downstream of Ihh or independently of Ihh signaling.