bluuebunny/crossref_metadata_2025 huggingface dataset:
Repeated maternal glucocorticoid administration and the developing liver in fetal sheep
Authors: Sloboda DM , Newnham JP ,   Challis
Published: December 2004

Prenatal glucocorticoid exposure has been associated with a reduction in birth weight and postnatal alterations in glucose homeostasis and hypothalamic-pituitary-adrenal (HPA) axis function. The mechanisms underlying these responses are unknown, although changes in fetal hepatic development may play an important role. The fetal liver produces key regulators of fuel metabolism and of the developing HPA axis that are altered by glucocorticoids. The local availability of glucocorticoids is regulated, in part, by corticosteroid-binding protein (CBG), glucocorticoid receptors (GR) and by the enzyme 11beta-hydroxysteroid dehydrogenase (11betaHSD), but the effects of maternal glucocorticoid administration on the expression of these genes in the fetal liver are unknown. 11betaHSD1 is the predominant form of this enzyme present in the liver and is responsible for the conversion of cortisone to cortisol. To determine if prenatal glucocorticoid exposure alters fetal hepatic regulation of CBG, 11betaHSD1 and GRs, we treated pregnant ewes with betamethasone (0.5 mg/kg) intramuscularly at 104, 111 and 118 days of gestation (term 150 days). Animals were killed at 125 or 146 days of gestation. Maternal betamethasone administration did not alter mean cord plasma glucose but significantly decreased cord plasma insulin levels (P<0.05) at 125 days of gestation. At 146 days of gestation, cord plasma glucose levels were significantly increased without alterations in insulin levels following maternal betamethasone treatment (P<0.05). Maternal betamethasone administration resulted in a significant increase in fetal hepatic 11betaHSD1 mRNA and protein levels at 125 days of gestation (P<0.05). CBG mRNA levels were significantly elevated over control at 125 days although levels of CBG protein were not significantly different. GR protein levels were not statistically different at either 125 or 146 days of gestation following glucocorticoid administration. These data suggest that prenatal betamethasone exposure in the ovine fetus results in alterations in cord glucose and insulin levels as well as alterations in hepatic 11betaHSD1 mRNA and protein expression. These changes in 11betaHSD1 increase the potential to generate local cortisol from circulating cortisone. We speculate that this could affect expression of glucocorticoid-dependent hepatic enzymes involved with the regulation of glucose production and HPA responsiveness.
High occurrence of thyroid multinodularity and low occurrence of subclinical hypothyroidism among tobacco smokers in a large population study
Authors: Knudsen N , Bulow I , Laurberg P , Perrild H , Ovesen L , Jorgensen T
Published: December 2004

Tobacco smoking increases the risk of goitre and Graves' disease, but the association with thyroid nodularity and hypothyroidism has not been settled. We investigated 4649 subjects from the general population with questionnaires, thyroid ultrasonography and blood tests. The results were analysed in multivariate regression models. Tobacco smoking was associated with an increased prevalence of thyroid multinodularity (odds ratio (OR) 1.9; 95% confidence interval (CI) 1.4-2.5), but not with increased prevalence of solitary thyroid nodules. The tendency was for a stronger association in the area with the most pronounced iodine deficiency (P for interaction=0.08). Lower levels of serum TSH were found among tobacco smokers (P<0.001), but this association disappeared when adjustment was made for thyroid nodularity and thyroid Volume. The prevalence of elevated TSH levels was markedly reduced among smokers (OR 0.47; 95% CI 0.33-0.67). No association was found between smoking and hyperthyroidism. The observed associations seem to be explainable by the blocking of iodine uptake and organification in the thyroid by thiocyanate, a degradation product of cyanide in tobacco smoke.
Evidence for a negative feedback in the control of eel growth hormone by thyroid hormones
Authors: Rousseau K , Le Belle N , Sbaihi M , Marchelidon J , Schmitz M , Dufour S
Published: December 2004

The regulation of growth hormone (GH) by thyroid hormones (THs) has been shown to present species variation. We investigated the regulation of GH in the eel, a representative of an ancient group of teleosts. In vivo administration of triiodothyronine (T(3)) or thyroxine (T(4)) significantly reduced pituitary and serum GH levels, as measured by homologous RIA. In order to investigate the ability of THs to regulate GH production directly at the pituitary level, we used a long-term, serum-free primary culture of eel pituitary cells. Both T(3) and T(4) inhibited GH release in a concentration-dependent manner, producing up to 50% inhibition at 10 nM, with an ED(50) of <0.2 nM, within the range of their physiological circulating levels. Other hormones also acting via the nuclear receptor superfamily, such as sex steroids (testosterone, estradiol and progesterone) and corticosteroid (cortisol), had no effect on GH release in vitro, underlining the specificity of the regulatory effect of THs on GH. Measurement of both GH release and cellular content for calculation of GH production in vitro indicated that THs not only inhibited GH release but also GH synthesis. Dot-blot assay of GH messenger RNA (mRNA) using an homologous eel cDNA probe showed a decrease in GH mRNA levels in cells cultured in the presence of T(3), as compared with control cells. This demonstrated that the inhibition of T(3) on GH synthesis was mediated by a decrease in GH mRNA steady state levels. In conclusion, we demonstrate inhibitory regulation of eel GH synthesis and release by THs, exerted directly at the pituitary level. These data contrast with the rat, where THs are known to have a stimulatory effect and suggest that the pattern observed here in an early vertebrate and also found in birds, reptiles and some mammals including humans, may represent an ancestral and more generalized vertebrate pattern of TH regulation of pituitary GH.
Adult growth hormone treatment reduces hypertension and obesity induced by an adverse prenatal environment
Authors: Vickers MH , Ikenasio BA , Breier BH
Published: December 2004

The discovery of a link between an adverse in utero environment and the propensity to develop metabolic and cardiovascular disease in adult life is one of the most important advances in epidemiological research of recent Years. Increasing experimental evidence suggests that alterations in the fetal environment may have long-term consequences for the development of metabolic disorders in adult life. This process has been termed 'fetal programming' and we have shown that undernutrition of the mother during gestation leads to development of the metabolic syndrome X during adult life. Striking metabolic similarities exist between syndrome X and untreated GH deficiency (GHD). In the present study we have investigated the effects of GH treatment on blood pressure and metabolic parameters. Virgin Wistar rats (age 75+/-5 days, n=20 per group) were time-mated and randomly assigned to receive food either ad libitum (AD) or 30% of AD intake (UN) throughout pregnancy. At weaning, male offspring were assigned to one of two diets (control or hypercaloric (30% fat)). Systolic blood pressure was measured at day 100 and following twice daily treatment with recombinant bovine GH for 21 days. GH treatment increased body weights in all treated animals but significantly reduced retroperitoneal and gonadal fat pad weights. Following GH treatment, systolic blood pressure was markedly decreased in all UN offspring. Saline-treated animals showed no change in systolic blood pressure over the treatment period. GH treatment increased heart-to-body weight ratio in all GH-treated animals. Our data demonstrated that GH treatment reduces hypertension and improves cardiovascular function in animals exposed to adverse environmental conditions during fetal or postnatal life.
Calcitonin decreases the adherence and survival of HEK-293 cells by a caspase-independent mechanism
Authors: Findlay DM , Raggatt LJ , Bouralexis S , Hay S , Atkins GJ , Evdokiou A
Published: December 2004

We recently reported that calcitonin (CT) can profoundly inhibit the growth of HEK-293 cells transfected with the human calcitonin receptor (hCTR). We also obtained preliminary evidence that suggested a role for CT in cell survival, and in the present study we have investigated the pro-apoptotic action of CT, which we observe in conditions of low serum concentration. Under these conditions, we have found that CT treatment of HEK-293 cells stably transfected with the insert-negative form of the human CTR (HR12 cells) caused a time-dependent decrease in cell number associated with loss of cellular attachment. Loss of cellular adherence in CT-treated cultures caused programmed cell death, as shown by Annexin V staining of cells, failure of cells to exclude Trypan Blue dye, condensation and cleavage of nuclear DNA, and appearance of hypodiploid cells in fluorescence-activated cell sorting (FACS) analysis. The accumulation of non-adherent cells and cell death was concomitant with increased intracellular activity of caspase-3. However, inhibition of caspase activation in HR12 cells did not prevent CT-mediated loss of attachment and did not maintain the viability of non-adherent cells, indicating that caspase activation accompanied, but was probably not the cause of, the loss of cell viability. Neither the effects of CT on cell survival nor the activation of caspase-3 were observed in serum-replete conditions, suggesting that serum-derived factors provide protection of cells from CT-induced apoptosis. The inhibitory effects of CT on cell growth were found previously to be related to activation of Erk1/2 MAP kinase. In the present experiments, it was found that the Erk1/2 inhibitor, PD 98059, inhibited the CT-induced loss of cellular adherence and the consequent reduction in cell numbers. These results demonstrate that CT can negatively affect cell survival and they identify roles for cell adherence and MAP kinase activation in this process.
Definition and characterization of relative hypo- and hyperleptinemia in a large Caucasian population
Authors: Buettner R , Bollheimer LC , Zietz B , Drobnik W , Lackner K , Schmitz G , Scholmerich J , Palitzsch KD
Published: December 2004

The adipocyte-derived hormone, leptin, has been implicated in the regulation of appetite, weight gain and glucose homeostasis as well as in liver fibrogenesis, hematopoiesis and immune function. No previous reports have clearly defined pathologically elevated or decreased serum leptin levels for Caucasian adults. The aim of this study was to define and characterize subjects with relative hyper- and hypoleptinemia in a large population-based German cohort. Percentiles of leptin levels by body mass index (BMI) were calculated from 4971 adult Germans, and the participants with leptin levels above the 95th and below the 5th percentile were defined as relatively hyperleptinemic and relatively hypoleptinemic, respectively, for their BMI. These participants were compared with the intermediate group with respect to anthropometric and clinical data and parameters of glucose and iron metabolism, lipid status, renal, adrenal and reproductive function. Relatively hyperleptinemic participants (HL) showed higher insulin, c-peptide, and total cholesterol levels than the hypoleptinemic subjects; in males, ferritin levels were higher and testosterone levels lower in the HL group. In conclusion, we report the first percentile curves for serum leptin by BMI in a large Caucasian population. Relatively low leptin values may be associated with a lower metabolic risk than relatively high serum leptin values.
Comparative peptidomics of the endocrine pancreas: islet hormones from the clawed frog Xenopus laevis and the red-bellied newt Cynops pyrrhogaster
Authors: Conlon JM , Kim JB , Johansson A , Kikuyama S
Published: December 2004

Electrospray mass spectrometry coupled with reverse-phase HPLC was used to identify peptides in the molecular mass range 3000-6000 Da in extracts of the pancreata of the clawed frog Xenopus laevis (Anura: Pipidae) and the red-bellied newt Cynops pyrrhogaster (Caudata: Salamandridae). Amino acid sequences of insulins, peptides derived from the post-translational processing of proglucagons and pancreatic polypeptide were determined by automated Edman degradation. Three molecular forms of insulin were isolated from the tetraploid organism X. laevis that represent insulin-1 and insulin-2, as deduced from the nucleotide sequences of previously characterized cDNAs, and a third form which differed from insulin-2 by the single amino acid substitution Asp(21)-->Glu in the B-chain. The amino acid sequence of Xenopus preproglucagons (genes 1 and 2 ) may be deduced from the nucleotide sequences of cDNAs but the pathways of post-translation processing of the precursors are not known. Two molecular forms of glucagon with 36 amino acids, derived from genes 1 and 2 and representing glucagon-29 extended from its C terminus by different heptapeptides, and five molecular forms of glucagon-like peptide 1 (GLP-1) were isolated. The GLPs represent proglucagon-(77-113), -(122-158) and -(160-191) from gene 1, and proglucagon-(77-113) and -(160-191) from gene 2. A single molecular form of insulin, glucagon-36, a C-terminally alpha-amidated GLP-1 with 30 amino acid residues, a 33 amino acid residue GLP-2 and pancreatic polypeptide were isolated from the pancreatic extract of the diploid organism C. pyrrhogaster. This study has illustrated the power of electrospray mass spectrometry for the rapid and reliable identification of peptides in chromatographic fractions without the need to use radioimmunoassay, radioreceptor assay or bioassay.
Modulation of the peroxisomal gene expression pattern by dehydroepiandrosterone and vitamin D: therapeutic implications
Authors: Depreter M , Vandesompele J , Espeel M , Speleman F , Roels F
Published: December 2004

Peroxisomes are ubiquitous organelles required for several metabolic functions. Their dysfunction is responsible for a group of human inherited disorders. In the search for endogenous factors regulating the peroxisomal compartment in normal liver, we treated female rats with dehydroepiandrosterone (DHEA) and 25-hydroxycholecalciferol for 1 and 6 days. Relative transcription levels of 39 selected genes were evaluated by real-time quantitative RT-PCR analysis. Catalase (peroxisomal marker)-specific activity was assayed in total liver homogenate and peroxisomes were visualized by catalase localization. DHEA induced peroxisome proliferation and raised catalase specific activity. Expression levels of 16 (of which 11 were peroxisomal) genes were altered. Pex 11, acyl-CoA oxidase,l - andd -multifunctional enzyme, thiolase 1, phytanoyl-CoA hydroxylase, 70 kDa peroxisomal membrane protein and very long chain acyl-CoA synthetase were upregulated, three others were downregulated. Vitamin D caused downregulation of six genes. Administration of vitamin D to peroxisomal disorder patients may be contraindicated. The adrenocortical hormone DHEA is a potential natural regulator of the peroxisomal compartment. Its therapeutic use in X-linked adrenoleukodystrophy, some other beta-oxidation defects and classical Refsum should be considered.
Protein tyrosine phosphatases are involved in LH/chorionic gonadotropin and 8Br-cAMP regulation of steroidogenesis and StAR protein levels in MA-10 Leydig cells
Authors: Paz C , Cornejo Maciel F , Maloberti P , Walsh LP , Stocco DM , Podesta EJ
Published: December 2004

The LH signal transduction pathway features the activation of protein tyrosine phosphatases (PTPs) as one of the components of a cascade that includes other well characterized events such as cAMP-dependent protein kinase A (PKA) activation. Moreover, the action of PTPs is required to increase the rate-limiting step in steroid biosynthesis, namely the cAMP-regulated transfer of cholesterol to the inner mitochondrial membrane. Since both PKA activity and steroidogenic acute regulatory (StAR) protein induction are obligatory steps in this transfer of cholesterol, the present study was performed to investigate the role of PTPs in the regulation of PKA activity and StAR expression in response to LH/chorionic gonadotropin (CG) and 8Br-cAMP in MA-10 cells. While the exposure of MA-10 cells to the PTP inhibitor, phenylarsine oxide (PAO), did not modify PKA activity, it partially inhibited the effect of human CG and cAMP analog on StAR protein levels. Time-course studies demonstrated that PAO inhibited cAMP induction of StAR protein and mRNA. At 30 min, the effect on cAMP-stimulated StAR protein levels was a 35% inhibition, progressing to up to 90% inhibition at 120 min of stimulation. The maximal inhibitory effect on cAMP-induced StAR mRNA level was obtained at 60 min (85%). In summary, these results demonstrate that inhibition of PTP activity affected both StAR protein and mRNA synthesis and suggest that the activity of hormone-regulated PTPs is a requirement in the LH signaling cascade that results in the up-regulation of StAR protein and, subsequently, increased steroid synthesis.
Pharmacokinetics, safety and tolerability of three dosage regimens of buccal adhesive testosterone tablets in healthy men suppressed with leuprorelin
Authors: Baisley KJ , Boyce MJ , Bukofzer S , Pradhan R , Warrington SJ
Published: December 2004

We used a randomised, double-blind, crossover design to evaluate the pharmacokinetics, safety and tolerability of three doses of buccal adhesive testosterone tablets (BATT). Twenty-four healthy men, whose endogenous testosterone was suppressed to </=5.38 nmol/l with leuprorelin acetate, took BATT (10, 20 or 30 mg) daily for 10 days. There was a 4-day washout between treatments. Substantial testosterone absorption occurred from BATT, and mean serum testosterone, free testosterone and dihydrotestosterone (DHT) concentrations over 24 h showed circadian variation. Steady state was reached by day 5. Average 24-h concentrations for the three BATT doses were within the normal range for eugonadal men: testosterone 11.67-14.57 nmol/l, free testosterone 0.026-0.33 nmol/l and DHT 1.66-2.03 nmol/l. On all three doses, peak testosterone and free testosterone was reached 8-9 h after tablet application; DHT peaked about 1-2 h later, and declined more slowly. Hormone concentrations increased with BATT dose, but increases were less than dose-proportional. There was no evidence of testosterone accumulation. BATT was well tolerated.
Residues in the C-terminal region of activin A determine specificity for follistatin and type II receptor binding
Authors: Fischer WH , Park M , Donaldson C , Wiater E , Vaughan J , Bilezikjian LM , Vale W
Published: December 2004

Activin is a secreted growth factor that signals by binding two related classes of single transmembrane receptors at the cell surface. The interaction of activin with its receptors is highly regulated by other cell surface receptors, antagonistic ligands, and high affinity extracellular binding proteins such as follistatin. Two activin A mutants, the deletion mutant des[85-109]-activin A and the point mutant K102E-activin A (K102E), were investigated with respect to their ability to bind cell surface receptors and the binding protein follistatin. The deletion mutant exhibits low affinity for both receptors and follistatin whereas the point mutant fails to bind cell surface receptors but binds follistatin-288 with high affinity. K102E is able to compete with wild type activin to bind to follistatin and can thus increase the concentration of activin available for receptor binding and signaling. These findings underline the importance of the C-terminal region of activin for binding interactions and show that different residues in this region are involved in cell surface receptor and follistatin interactions.
Activation of the MAP kinase pathway induces chicken ovalbumin upstream promoter-transcription factor II (COUP-TFII) expression in human breast cancer cell lines
Authors: More E , Fellner T , Doppelmayr H , Hauser-Kronberger C , Dandachi N , Obrist P , Sandhofer F , Paulweber B
Published: December 2004

Growth factors are essential for cellular growth and differentiation in both normal and malignant human breast epithelial cells. In the present study we investigated the effect of epidermal growth factor (EGF), transforming growth factor alpha (TGFalpha) and phorbol myristate acetate (PMA) on chicken ovalbumin upstream promoter-transcription factor (COUP-TF) expression in human breast cancer cells. The orphan receptors COUP-TFI and COUP-TFII are members of the nuclear receptor superfamily. The high degree of evolutionary conservation of these proteins strongly argues for an important biological function. COUP-TF expression was highest in SK-BR3 cells (approximately 130 amol/ micro g total RNA), while the lowest COUP-TF expression was observed in MCF-7 cells (3.5 amol/ micro g total RNA). While treatment of EGF, TGFalpha and PMA induced expression of COUP-TFII, COUP-TFI did not respond to these agents. Oncostatin M (OSM) is known to exert an antiproliferative effect in breast cancer cells. Treatment of MCF-7 cells with OSM resulted in an approximately 90% reduction of COUP-TFII mRNA expression. In SK-BR3 cells, treatment with the MEK inhibitor UO126 resulted in a profound suppression of endogenous COUP-TFII expression. Furthermore, cotreatment with UO126 prevented induction of COUP-TFII expression by EGF in MCF-7 cells. In conclusion, our data provide evidence, for the first time, that mitogenic substances which activate the MAP kinase pathway, can induce COUP-TFII expression. Our results strongly suggest that an active MAP kinase pathway is essential for COUP-TFII expression in human breast cancer cells.
Neurohypophysial hormone regulation of Cl- secretion: physiological evidence for V1-type receptors in sea bass gill respiratory cells in culture
Authors: Guibbolini ME , Avella M
Published: December 2004

Neurohypophysial hormone receptors were studied in primary cultures of sea bass (Dicentrarchus labrax) gill respiratory-like cells grown on permeable supports. This preparation was previously shown to provide a functional model for investigating the hormonal regulation of Cl- secretion. Under control conditions, the cultured monolayered epithelium had a short-circuit current (ISC) of 3.5+/-1.1 micro A x cm(-2). This current had previously been identified as an active Cl- secretion. The addition of increasing concentrations of the fish neurohypophysial hormones, arginine vasotocin (AVT) or isotocin (IT), elicited a concentration-dependent stimulation of the ISC. Maximal increases of 60.9+/-12.1% and 117.7+/-28.0% above the basal ISC value were obtained for 10(-7) M AVT and IT respectively. Half-maximal effects were obtained for 3.1 x 10(-9) M AVT and for 1.4 x 10(-9) M IT. Mucosal application of 1.0 mM diphenylalamine-2-carboxylic acid (a specific blocker of Cl- channels) after serosal addition of 5 x 10(-8) M AVT or IT inhibited not only the basal but also the stimulated current, revealing a correlation with a hormone-dependent Cl- transport. Specific V1 or V2 receptor analogues of vasopressin (mammalian hormone) were used to characterize the type of neurohypophysial hormone receptors pharmacologically. While the V1 agonist [Phe2,Orn8]-oxytocin stimulated the basal Cl- secretion with a similar profile to that of AVT or IT, the V2 agonist [Deamino1,Val4,d -Arg8]-vasopressin had no effect. The V1 antagonist [d(CH2)5 1,O-Me-Tyr2,Arg8]-vasopressin used at a concentration of 5 x 10(-7) M totally reversed the 10-8 M AVT-stimulated Cl- secretion, whereas the V2 antagonist [d(CH2)5 1,d -Ile2,Ile4,Arg8,Ala9]-vasopressin used at the same concentration had no significant effect. In contrast, similar experiments carried out in the presence of 10(-8) M IT showed that both antagonists significantly reduced the IT-stimulated Cl- secretion, with the efficiency of the V1 receptor antagonist being significantly greater than that of the V2. This study provides evidence for neurohypophysial hormone control of Cl- secretion in fish cultured gill respiratory cells. It suggests that on a physiological basis the hormonal effect is shared by the two peptides present in fish neurohypophysis (AVT and IT), acting by means of two distinct, although pharmacologically similar, V1-type receptors (according to the mammalian classification). These specific receptors are expected to play an important role in controlling ion homeostasis in seawater fish.
Establishment of T cell lines to bovine beta-casein and beta-casein-derived epitopes in patients with type 1 diabetes
Authors: Monetini L , Barone F , Stefanini L , Petrone A , Walk T , Jung G , Thorpe R , Pozzilli P , Cavallo MG
Published: December 2004

Enhanced cellular immune response to bovine beta-casein has been reported in patients with type 1 diabetes. In this study we aimed to establish beta-casein-specific T cell lines from newly diagnosed type 1 diabetic patients and to characterise these cell lines in terms of phenotype and epitope specificity. Furthermore, since sequence homologies exist between beta-casein and putative beta-cell autoantigens, reactivity to the latter was also investigated. T cell lines were generated from the peripheral blood of nine recent onset type 1 diabetic patients with different HLA-DQ and -DR genotypes, after stimulation with antigen pulsed autologous irradiated antigen presenting cells (APCs) and recombinant human interleukin-2 (rhIL-2). T cell line reactivity was evaluated in response to bovine beta-casein, to 18 overlapping peptides encompassing the whole sequence of beta-casein and to beta-cell antigens, including the human insulinoma cell line, CM, and a peptide from the beta-cell glucose transporter, GLUT-2. T cell lines specific to beta-casein could not be isolated from HLA-matched and -unmatched control subjects. beta-Casein T cell lines reacted to different sequences of the protein, however a higher frequency of T cell reactivity was observed towards the C-terminal portion (peptides B05-14, and B05-17 in 5/9 and 4/9 T cell lines respectively). Furthermore, we found that 1 out of 9 beta-casein-specific T cell lines reacted also to the homologous peptide from GLUT-2, and that 3 out of 4 of tested cell lines reacted also to extracts of the human insulinoma cell line, CM. We conclude that T cell lines specific to bovine beta-casein can be isolated from the peripheral blood of patients with type 1 diabetes; these cell lines react with multiple and different sequences of the protein particularly towards the C-terminal portion. In addition, reactivity of beta-casein T cell lines to human insulinoma extracts and GLUT-2 peptide was detected, suggesting that the potential cross-reactivity with beta-cell antigens deserves further investigation.
Evaluation of relative roles of LH and FSH in regulation of differentiation of Leydig cells using an ethane 1,2-dimethylsulfonate-treated adult rat model
Authors: Sriraman V , Sairam MR , Rao AJ
Published: December 2004

The relative role of LH and FSH in regulation of differentiation of Leydig cells was assessed using an ethane 1,2-dimethylsulfonate (EDS)-treated rat model in which endogenous LH or FSH was neutralized from day 3 to day 22 following EDS treatment. Serum testosterone and the in vitro response of the purified Leydig cells to human chorionic gonadotropin (hCG) was monitored. In addition RNA was isolated from the Leydig cells to monitor the steady-state mRNA levels by RT-PCR for 17alpha-hydroxylase, side chain cleavage enzyme, steroidogenic acute regulatory protein (StAR), LH receptor, estrogen receptor (ER-alpha) and cyclophilin (internal control). Serum testosterone was undetected and the isolated Leydig cells secreted negligible amount of testosterone on stimulation with hCG in the group of rats that were treated with LH antiserum following EDS treatment. RT-PCR analysis revealed the absence of message for cholesterol side chain cleavage enzyme and 17alpha-hydroxylase although ER-alpha and LH receptor mRNA could be detected, indicating the presence of undifferentiated precursor Leydig cells. In contrast, the effects following deprival of endogenous FSH were not as drastic as seen following LH neutralization. Deprival of endogenous FSH in EDS-treated rats led to a significant decrease in serum testosterone and in vitro response to hCG by the Leydig cells. Also, there was a significant decrease in the steady-state mRNA levels of 17alpha-hydroxylase, cholesterol side chain cleavage enzyme, LH receptor and StAR as assessed by a semiquantitative RT-PCR. These results establish that while LH is obligatory for the functional differentiation of Leydig cells, repopulation of precursor Leydig cells is independent of LH, and also unequivocally establish an important role for FSH in regulation of Leydig cell function.
Fusion pore or porosome: structure and dynamics
Authors: Jena BP
Published: December 2004

Electrophysiological measurements on live secretory cells almost a decade ago suggested the presence of fusion pores at the cell plasma membrane. Membrane-bound secretory vesicles were hypothesized to dock and fuse at these sites, to release their contents. Our studies using atomic force microscopy on live exocrine and neuroendocrine cells demonstrate the presence of such plasma membrane pores, revealing their morphology and dynamics at near nm resolution and in real time.
Effects of cortisol and oestradiol on hepatic 11beta-hydroxysteroid dehydrogenase type 1 and glucocorticoid receptor proteins in late-gestation sheep fetus
Authors: Gupta S , Alfaidy N , Holloway AC , Whittle WL , Lye SJ , Gibb W ,   Challis
Published: December 2004

In the late-gestation sheep, increased fetal plasma cortisol concentration and placental oestradiol (E(2)) output contribute to fetal organ maturation, in addition to the onset of parturition. Both cortisol and E(2) are believed to regulate the enzyme 11beta-hydroxysteroid dehydrogenase type 1 (11beta-HSD1), which interconverts bioactive 11-hydroxy glucocorticoids and their inactive 11-keto metabolites. 11beta-HSD1, abundantly expressed in fetal liver, operates primarily as a reductase enzyme to produce bioactive cortisol and thus regulates local hepatic glucocorticoid concentrations. Cortisol acts through the glucocorticoid receptor (GR) present in the liver. In this study, we examined the effects of cortisol and E(2) on hepatic 11beta-HSD1 and GR in the liver of chronically catheterized sheep fetuses treated with saline (n=5), cortisol (1.35 mg/h; n=5), saline+4-hydroxyandrostendione, a P450 aromatase inhibitor (4-OHA; 1.44 mg/h; n=5), or cortisol+4-OHA (n=5). Cortisol infusion resulted in increased plasma concentrations of fetal cortisol and E(2); concurrent administration of 4-OHA attenuated the increase in plasma E(2) concentrations. Using immunohistochemistry, we showed that fetal hepatocytes expressed both 11beta-HSD1 and GR proteins. Cortisol treatment increased GR in both cytosol and nuclei of hepatocytes; concurrent administration of 4-OHA was associated with distinct nuclear GR staining. Western blot revealed that cortisol, in the absence of increased E(2) concentrations, significantly increased concentrations of 11beta-HSD1 (34 kDa) and GR (95 kDa) proteins. 11beta-HSD1 enzyme activity was measured in the liver microsomal fraction in the presence of [(3)H]cortisone (10(-)(6) M) or [(3)H]cortisol (10(-)(6) M) and NADPH (reductase activity) or NADP(+) (dehydrogenase activity) respectively. 11beta-HSD1 reductase activity was significantly greater in the presence of cortisol. In summary, we found that, in sheep during late gestation, cortisol increased both 11beta-HSD1 and GR in the fetal liver, and these effects were accentuated in the absence of increased E(2).
Effect of recombinant porcine IGF-binding protein-3 on proliferation of embryonic porcine myogenic cell cultures in the presence and absence of IGF-I
Authors: Pampusch MS , Kamanga-Sollo E , White ME , Hathaway MR , Dayton WR
Published: December 2004

IGF-binding protein (IGFBP)-3 is produced by cultured porcine embryonic myogenic cell (PEMC) cultures and is secreted into the medium. Levels of secreted IGFBP-3 and IGFBP-3 mRNA are significantly reduced during differentiation and increase after differentiation is complete, suggesting that IGFBP-3 may play some role in myogenesis and/or in changes in myogenic cell proliferation that accompany differentiation. IGFBP-3 reportedly may either suppress or stimulate proliferation of cultured cells depending on cell type. Additionally, IGFBP-3 has been shown to affect proliferation via both IGF-dependent and IGF-independent mechanisms in some cell types but not all. Currently, the effect, if any, of IGFBP-3 on myogenic cell proliferation is not known. Consequently, the goal of this study was to assess the IGF-I-dependent and IGF-I-independent actions of recombinant porcine IGFBP-3 on proliferation of cultured porcine myogenic cells. To facilitate these investigations, we have expressed porcine IGFBP-3 in the baculovirus system, purified and characterized the expressed recombinant porcine IGFBP-3 (rpIGFBP-3), and produced and characterized an anti-porcine IGFBP-3 antibody that neutralizes the biological activity of porcine IGFBP-3. rpIGFBP-3 suppressed IGF-I-stimulated proliferation of PEMCs in a concentration-dependent manner with equimolar concentrations of IGF-I and rpIGFBP-3, resulting in complete suppression of IGF-I-stimulated proliferation. rpIGFBP-3 also suppressed Long-R3-IGF-I-stimulated proliferation of PEMC, indicating that rpIGFBP-3 possesses IGF-independent activity in this cell system. These data have established that IGFBP-3 has the potential to affect proliferation of PEMCs during critical periods of muscle development that may impact ultimate muscle mass achievable postnatally.
Expression and role of mannose receptor/terminal high-mannose type oligosaccharide on osteoclast precursors during osteoclast formation
Authors: Morishima S , Morita I , Tokushima T , Kawashima H , Miyasaka M , Omura K , Murota S
Published: December 2004

Osteoclasts are formed from hematopoietic precursors via cell-cell fusion. We have previously reported that mannose residues are expressed on the outer membranes of monocytes during osteoclast differentiation. In the present study, we have attempted to demonstrate the pattern of expression levels of terminal high-mannose type oligosaccharide and to show that the mannose receptor is expressed on osteoclast precursor cells. Osteoclasts were formed using three different systems, namely mouse bone marrow cell culture, co-culture of mouse spleen cells with stromal cells, and RAW264.7 cell cultures. During osteoclast differentiation, the expression of terminal high-mannose type oligosaccharide gradually increased and then peaked at the stage of fusion in all three systems. Expression of the mannose receptor gradually increased during osteoclast differentiation in bone marrow cells and the co-culture system. In contrast, that in RAW264.7 cells had already been detected in the absence of the soluble receptor activator of NF-kappaB ligand and did not change during osteoclast differentiation. To ascertain whether expression of high-mannose type oligosaccharide is involved in tartrate-resistant acid phosphatase (TRAP)-positive multinucleated cell (MNC) formation, glycosidase inhibitors were used on RAW264.7 cell culture. Castanospermine, an inhibitor of glucosidase I, inhibited the TRAP-positive MNCs, and deoxymannojirimycin, an inhibitor of alpha-mannosidase I, increased the TRAP-positive MNC formation. These results indicate that the binding of terminal high-mannose and mannose receptor is important for the process of cellular fusion in osteoclast formation.
Hypothyroidism alters mitochondrial morphology and induces release of apoptogenic proteins during rat cerebellar development
Authors: Singh R , Upadhyay G , Godbole MM
Published: December 2004

Thyroid hormone (TH) deficiency leads to extensive apoptosis during cerebellar development, but the mechanism still remains unclear. Different signals also converge on mitochondria during apoptosis to induce the release of apoptogenic proteins that activate proteolytic cascade through specific enzymes called caspases. Here we studied the effect of hypothyroidism on alterations in mitochondrial structure and translocation of apoptogenic molecules during rat cerebellar development. Structural analysis of mitochondria was studied by electron microscopy. The translocation of apoptogenic molecules was analyzed by Western blotting. TH deficiency led to vacuolization, enlargement and decrease in the number of cristae. The majority of the proapoptotic molecule, Bax, was localized in mitochondria under hypothyroid conditions whereas a limited presence of Bax was detected in the euthyroid state. Translocation of cytochrome c, apoptosis-inducing factor (AIF) and second mitochondrial-derived activator of caspases (SMAC) from mitochondria to cytosol was detected primarily in early developmental stages in the hypothyroid condition. These experimental results demonstrate that TH maintains mitochondrial architecture and inhibits the release of apoptogenic molecules to prevent excess apoptosis during cerebellar development.
Growth retardation induced by dexamethasone is associated with increased apoptosis of the growth plate chondrocytes
Authors: Chrysis D , Ritzen EM , Savendahl L
Published: December 2004

Glucocorticoids cause significant growth retardation in mammals and humans and decreased proliferation of chondrocytes has been considered as the main local mechanism. Death by apoptosis is an important regulator of homeostasis in multicellular organisms. Here we chose to study the role of apoptosis in growth retardation caused by glucocorticoid treatment. We treated 7-week-old male rats with dexamethasone (5 mg/kg/day) for 7 days. Apoptosis was studied in tibiae growth plates by the TUNEL method. Immunoreactivity for parathyroid hormone-related peptide (PTHrP), caspase-3, and the anti-apoptotic proteins Bcl-2 and Bcl-x was also studied. Apoptosis was mainly localized in terminal hypertropic chondrocytes (THCs) in both control and dexamethasone-treated animals. Dexamethasone caused an increase in apoptosis which was fourfold in THCs (2.45+/-0.12 vs 0.62+/-0.09 apoptotic cells/mm growth plate, P<0.001), and 18-fold in proliferative chondrocytes (0.18+/-0.04 vs 0.01+/-0.007 apoptotic cells/mm growth plate, P<0.001). Increased apoptosis after dexamethasone treatment was accompanied by increased immunoreactivity for caspase-3 and decreased immunoreactivity for the anti-apoptotic proteins Bcl-2 and Bcl-x, which further supports our apoptosis results. Dexamethasone also decreased the immunoreactivity for PTHrP, suggesting a role in the mechanism by which glucocorticoids induce apoptosis in the growth plate. We conclude that apoptosis is one mechanism involved in growth retardation induced by glucocorticoids. Premature loss of resting/proliferative chondrocytes by apoptosis could contribute to incomplete catch-up seen after prolonged glucocorticoid treatment.
Vitamin K stimulates osteoblastogenesis and inhibits osteoclastogenesis in human bone marrow cell culture
Authors: Koshihara Y , Hoshi K , Okawara R , Ishibashi H , Yamamoto S
Published: December 2004

Accumulating evidence indicates that menaquinone-4 (MK-4), a vitamin K(2) with four isoprene units, inhibits osteoclastogenesis in murine bone marrow culture, but the reason for this inhibition is not yet clear, especially in human bone marrow culture. To clarify the inhibitory mechanism, we investigated the differentiation of colony-forming-unit fibroblasts (CFU-Fs) and osteoclasts in human bone marrow culture, to learn whether the enhancement of the differentiation of CFU-Fs from progenitor cells might relate to inhibition of osteoclast formation. Human bone marrow cells were grown in alpha-minimal essential medium with horse serum in the presence of MK-4 until adherent cells formed colonies (CFU-Fs). Colonies that stained positive for alkaline phosphatase activity (CFU-F/ALP(+)) were considered to have osteogenic potential. MK-4 stimulated the number of CFU-F/ALP(+) colonies in the presence or absence of dexamethasone. The stimulation was also seen in vitamin K(1) treatment. These cells had the ability to mineralize in the presence of alpha-glycerophosphate. In contrast, both MK-4 and vitamin K(1) inhibited 1,25 dihydroxyvitamin D(3)-induced osteoclast formation and increased stromal cell formation in human bone marrow culture. These stromal cells expressed ALP and Cbfa1. Moreover, both types of vitamin K treatment decreased the expression of receptor activator of nuclear factor kappaB ligand/osteoclast differentiation factor (RANKL/ODF) and enhanced the expression of osteoprotegerin/osteoclast inhibitory factor (OPG/OCIF) in the stromal cells. The effective concentrations were 1.0 microM and 10 microM for the expression of RANKL/ODF and OPG/OCIF respectively. Vitamin K might stimulate osteoblastogenesis in bone marrow cells, regulating osteoclastogenesis through the expression of RANKL/ODF more than through that of OPG/OCIF.
Effects of the Japanese herbal medicine Keishi-bukuryo-gan and 17beta-estradiol on calcitonin gene-related peptide-induced elevation of skin temperature in ovariectomized rats
Authors: Noguchi M , Ikarashi Y , Yuzurihara M , Kase Y , Chen JT , Takeda S , Aburada M , Ishige A
Published: December 2004

The effects of a Japanese herbal medicine, Keishi-bukuryo-gan, and 17beta-estradiol on calcitonin gene-related peptide (CGRP)-induced elevation of skin temperature were investigated in ovariectomized (OVX) rats. Ovariectomy not only potentiated CGRP-induced elevation of skin temperature and arterial vasorelaxation but also induced a lower concentration of endogenous CGRP in plasma and up-regulation of arterial CGRP receptors, suggesting that lowered CGRP in plasma due to ovarian hormone deficiency increases the number of CGRP receptors and consequently amplifies the stimulatory effects of CGRP to elevate skin temperature. Oral Keishi-bukuryo-gan (100-1000 mg/kg, once a day for 7 days) restored a series of CGRP-related responses observed in OVX rats by normalizing plasma CGRP levels in a dose-dependent manner as effectively as s.c. injection. 17Beta-estradiol (0.010 mg/kg, once a day for 7 days). However, Keishi-bukuryo-gan did not affect the lower concentration of plasma estradiol and the decreased uterine weight due to ovariectomy, although the hormone replacement of 17beta-estradiol restored them. These results suggest that Keishi-bukuryo-gan, which does not confer estrogen activity on plasma, may be useful for the treatment of hot flashes in patients for whom estrogen replacement therapy is contraindicated, as well as menopausal women.
Differential regulation of rat testicular 5alpha-reductase type 1 and 2 isoforms by testosterone and FSH
Authors: Pratis K , O'Donnell L , Ooi GT , Stanton PG , McLachlan RI , Robertson DM
Published: December 2004

Testosterone is metabolised to the more potent androgen, dihydrotestosterone, by the 5alpha-reductase (5alphaR) enzyme. We previously showed that 5alpha-reduced androgens are important for maintaining androgen action on rat spermatogenesis when testicular testosterone concentrations are reduced. This study investigated expression and activity of the 5alphaR isoforms, type 1 (5alphaR-1) and type 2 (5alphaR-2), in the rat during hormone manipulation in order to understand the factors that regulate the testicular concentration of 5alphaR and testicular 5alpha-reduced androgen biosynthesis. Testicular 5alphaR-1 and 5alphaR-2 mRNA and enzyme activity were measured by real-time PCR and specific enzyme assays respectively. Hormone levels were first suppressed using two models of gonadotrophin suppression: testosterone and oestradiol treatment (LH/testosterone deficiency) or GnRH immunisation (LH/testosterone and FSH deficiency). Hormones were then either restored or suppressed for 6 days by a variety of hormonal treatments. 5alphaR-1 mRNA and enzyme activity increased when testosterone was suppressed, yet restoration of testosterone decreased 5alphaR-1 mRNA and enzyme activity, suggesting that testosterone negatively regulates 5alphaR-1. suppression of FSH decreased 5alphaR-1 mRNA yet FSH administration increased 5alphaR-1 mRNA, but no changes in 5alphaR-1 activity were observed within the 6 day period. In contrast to 5alphaR-1, testosterone did not affect the testicular concentration of 5alphaR-2 mRNA or activity, but there was evidence for modulation of 5alphaR-2 activity by FSH. Measurement of testicular androgens revealed that 5alphaR-1 was primarily responsible for the production of 5alpha-reduced metabolites. It is concluded that the 5alphaR isoforms in rat testis are differentially regulated by testosterone and FSH: testosterone negatively regulated 5alphaR-1 mRNA and enzyme activity but had no affect on 5alphaR-2, whereas FSH positively regulated 5alphaR-1 mRNA and appeared to regulate 5alphaR-2.
Prolonged underfeeding of sheep increases myostatin and myogenic regulatory factor Myf-5 in skeletal muscle while IGF-I and myogenin are repressed
Authors: Jeanplong F , Bass JJ , Smith HK , Kirk SP , Kambadur R , Sharma M , Oldham JM
Published: December 2004

The IGF axis is nutritionally sensitive in vivo and IGFs stimulate myoblast proliferation and differentiation in vitro, while myostatin inhibits these processes in vitro. We hypothesised that underfeeding would reversibly inhibit the myogenic activity of satellite cells in vivo together with decreased IGF-I and increased myostatin in muscle. Satellite cell activity was measured indirectly from the expression of proliferating cell nuclear antigen (PCNA) and the myogenic regulatory factors (MRFs), MyoD, Myf-5 and myogenin. Young sheep were underfed (30% of maintenance) and some killed after 1, 4, 12, 17, 21 and 22 weeks. Remaining underfed animals were then re-fed a control ration of pellets and killed after 2 days, and 1, 6 and 30 weeks. Expression of PCNA and MRFs decreased during the first week of underfeeding. This coincided with reduced IGF-I and myostatin mRNA, and processed myostatin. Subsequently, Myf-5, MyoD, myostatin mRNA and processed myostatin increased, suggesting that satellite cells may have become progressively quiescent. Long-term underfeeding caused muscle necrosis in some animals and IGF-I and MRF expression was increased in these, indicating the activation of satellite cells for muscle repair. Re-feeding initiated rapid muscle growth and increased expression of PCNA, IGF-I and the MRFs concurrently with decreased myostatin proteins. In conclusion, these data indicate that IGF-I and myostatin may work in a coordinated manner to regulate the proliferation, differentiation and quiescence of satellite cells in vivo.
Integration of mammalian circadian clock signals: from molecule to behavior
Authors: Okamura H
Published: December 2004

The core oscillatory mechanism consisting of gene transcription and translation is a unique feature and the astonishing discovery in circadian biology is that the rhythm of gene transcription reflects the behavioral rhythm almost perfectly. This means that the clock gene oscillation generated by the core loop in each suprachiasmatic nucleus neuron is coupled and amplified, and harmonized strongly so that oscillating activities are spread into the whole brain and to all those peripheral organs which contain peripheral clocks. Additionally, circadian changes are induced in behavior and hormone secretion. Investigations of biological clocks open the fascinating perspective to analyze the integrational mechanism of 'time', providing a bridge between single genes and the living organism as a whole.