bluuebunny/crossref_metadata_2025_split huggingface dataset:
Expression of a Leptin Receptor in Immortalized Gonadotropin-Releasing Hormone-Secreting Neurons*
Authors: Magni Paolo , Vettor Roberto , Pagano Claudio , Calcagno Alessandra , Beretta Elena , Messi Elio , Zanisi Mariarosa , Martini Luciano , Motta Marcella 
Published: January 2014

Abstract
Leptin is secreted by adipocytes and regulates food intake and energy balance through the activation of specific receptors (OB-R). Recent evidence suggests that it is also involved in the control of reproductive processes, by possibly acting on central and peripheral targets. In particular, it has been shown that leptin may indirectly stimulate GnRH release from hypothalamic fragments by acting on interneurons impinging on GnRH-secreting neurons. The possibility that leptin might additionally modulate the activity of GnRH-secreting neurons in a direct way has been addressed in the present study, by using the immortalized GnRH-secreting cell line GT1–7. The presence of OB-R messenger RNA (mRNA) (long form) was detected by RT-PCR analysis of total RNA from GT1–7 cells. An OB-R protein is also expressed in these cells, as shown by immunocytochemistry and by Western blot analysis. The latter has revealed the presence of a single immunoreactive OB-R with an approximate size of 130 kDa. To study the functionality of these receptors, the effect of leptin treatment on GnRH secretion and gene expression in GT1–7 cells were evaluated. Under static conditions, GnRH release was stimulated by exposure to low concentrations of leptin (10−12m after 30 min; 10−10m after 60 min). The 10−12m dose was selected for studying the effect of leptin on GnRH secretion under dynamic conditions. To this purpose, GT1–7 cells were placed in a perifusion system; treatment with leptin (10−12m) for 60 min stimulated GnRH release with no changes of pulse frequency. On the contrary, exposure to leptin (10−12–10−10m) for 1, 3, 6, and 24 h did not affect GnRH gene expression in GT1–7 cells. The present results indicate that GT1–7 cells possess OB-Rs and that leptin may directly affect their function. Taken together with the available reports, these findings suggest that leptin might participate in the regulation of reproductive processes by acting at multiple levels, both centrally and peripherally.
Characterization of a Region of the Lutropin Receptor Extracellular Domain Near Transmembrane Helix 1 That Is Important in Ligand-Mediated Signaling*
Authors: Alvarez Carlos A. , Narayan Prema , Huang Jianing , Puett David 
Published: January 2014

AbstractThe lutropin receptor (LHR), a member of the G protein-coupled receptor family, contains a relatively large N-terminal extracellular domain, accounting for about half of the receptor and responsible for high affinity ligand binding, and a standard heptahelical portion with connecting loops and a C-terminal tail. LHR and the other two glycoprotein hormone receptors, i.e. the follitropin and TSH receptors, contain an invariant 10-amino acid residue sequence, FNPCEDIMGY (residues 328–337 in rat LHR), in the extracellular domain separated by only a few amino acid residues from the beginning of transmembrane helix 1. In view of the invariant nature of this region in the three glycoprotein hormone receptors and preliminary data in the literature on the importance of Glu332 and Asp333 in signal transduction, we undertook a systematic investigation of all 10 amino acid residues because this region may function as a switch or trigger for communicating ligand binding to the extracellular domain with a conformational change of the membrane-embedded C-terminal half of the receptor to activate G proteins, particularly Gs. A total of 36 single, double, and multiple replacements, as well as two deletions, of LHR were prepared and characterized in transiently transfected COS-7 cells. Of these mutants LHRs, 26 expressed on the cell surface in sufficient numbers that quantitative assessments could be made of human choriogonadotropin binding and ligand-mediated cAMP production. Replacements of Cys331 abolished ligand binding to intact cells, although binding could be detected after solubilization of the cells. Replacements of the other nine amino acid residues that did not interfere with receptor folding or trafficking had no significant effect on ligand binding affinity; however, replacements of Pro330, Glu332, and Asp333 resulted in diminished signaling, especially for the two acidic residues. An interesting observation was made in which replacement of Tyr337 with Ala or Asp, while having no profound change on receptor function, could overcome to some extent limited expression of replacements at positions 332 and/or 333, thus permitting a more definitive analysis of signaling. Replacement of the decapeptide sequence with Gly10 prevents expression, whereas deletion of all 10 residues and deletion of Glu332-Asp333 prevents functional expression at the cell surface. Thus, this invariant sequence in the glycoprotein hormones is required for proper folding, trafficking, and ligand-mediated signaling, but not ligand binding, in LHR. Amino acid residues, Glu332, Asp333, and to a limited extent, Pro330, are important in ligand-mediated signaling but not ligand binding.
Connective Tissue Growth Factor (IGFBP-rP2) Expression and Regulation in Cultured Bovine Endothelial Cells*
Authors: Boes Mary , Dake Brian L. , Booth Barbara A. , Erondu Ngozi E. , Oh Youngman , Hwa Vivian , Rosenfeld Ron , Bar Robert S. 
Published: January 2014

Abstract
Media from large vessel endothelial cells (pulmonary artery, aorta) contained intact connective tissue growth factor (CTGF) and a dominant 19-kDa band. N-terminal analysis of the 19-kDa band showed sequence corresponding to CTGF amino acid 181–190, suggesting that the 19-kDa band represented a proteolytic fragment of CTGF. Intact CTGF was increased by cAMP but not by transforming growth factor-β (TGFβ). CTGF messenger RNA (mRNA) was not changed by cAMP nor TGFβ. In two microvessel endothelial cells, mRNA was found at low levels by PCR and Northern analysis, but no CTGF protein was seen on Western analysis. In the microvessel cells, TGFβ increased and cAMP did not change CTGF mRNA levels, with neither TGFβ nor cAMP increasing CTGF protein. The discordance between protein and mRNA levels in large vessel and microvessel endothelial cells was mostly explained by the effects of cAMP and TGFβ on media proteolytic activity; in large vessel cells, cAMP inhibited degradation of CTGF, whereas in microvessel cells, TGFβ and cAMP stimulated proteolytic activity against CTGF.
We conclude that in large vessel endothelial cells, cAMP increased intact CTGF protein by inhibiting degradation of CTGF, whereas TGFβ stimulated neither CTGF mRNA nor protein; in microvessel cells, TGFβ increased CTGF mRNA, while both TGFβ and cAMP stimulated CTGF degradation.
Transcriptional Activation of Insulin-Like Growth Factor-Binding Protein-4 by 17β-Estradiol in MCF-7 Cells: Role of Estrogen Receptor-Sp1 Complexes*
Authors: Qin Chunhua , Singh Pomila , Safe Stephen 
Published: January 2014

AbstractInsulin-like growth factor-binding protein-4 (IGFBP-4) is expressed in MCF-7 human breast cancer cells, and treatment of these cells with 17β-estradiol (E2) resulted in induction of IGFBP-4 gene expression (&gt;3-fold) and protein secretion (&gt;6-fold). To identify genomic sequences associated with E2 responsiveness, the 5′-promoter region (−1214 to +18) of the IGFBP-4 gene was cloned into a vector upstream from the firefly luciferase reporter gene, and E2 induced a 10-fold increase in luciferase activity in MCF-7 cells transiently transfected with this construct. Deletion analysis of this region of the IGFBP-4 gene promoter identified two GC-rich sequences at −559 to −553 and −72 to −64 that were important for E2-induced trans-activation. Gel mobility shift assays using 32P-labeled −569 to −540 and −83 to −54 oligonucleotides from the IGFBP-4 gene promoter showed that Sp1 protein bound these oligonucleotides to form a retarded band, and the intensity of the band was competitively decreased after coincubation with unlabeled IGFBP-4-derived and consensus Sp1 oligonucleotides. Mutation of the GC-rich sites within these sequences resulted in loss of the retarded band formation. Wild-type human estrogen receptor did not bind directly to the IGFBP-4 oligonucleotides; however, human estrogen receptor enhanced Sp1-DNA binding in a concentration-dependent manner. The results of this study demonstrate that at least two GC-rich sequences at −559 to −553 and− 72 to −64 are required for induction of IGFBP-4 gene expression by E2 in MCF-7 cells.
Mitogen-Activated Protein Kinase-Dependent Stimulation of Proliferation of Rat Lactotrophs in Culture by 3′,5′-Cyclic Adenosine Monophosphate*
Authors: Suzuki Shinichi , Yamamoto Isao , Arita Jun 
Published: January 2014

AbstractIntracellular cAMP regulates cell proliferation as a second messenger of extracellular signals in a number of cell types. We investigated, by pharmacological means, whether an increase in intracellular cAMP levels changes proliferation rates of lactotrophs in primary culture, whether there are interactions between signal transduction pathways of cAMP and the growth factor insulin, and where the dopamine receptor agonist bromocriptine acts in the cAMP pathway to inhibit lactotroph proliferation. Rat anterior pituitary cells, cultured in serum-free medium, were treated with cAMP-increasing agents, followed by 5-bromo-2′-deoxyuridine (BrdU) to label proliferating pituitary cells. BrdU-labeling indices indicative of the proliferation rate of lactotrophs were determined by double immunofluorescence staining for PRL and BrdU. Treatment with forskolin (an adenylate cyclase activator) or (Bu)2cAMP (a membrane-permeable cAMP analog) increased BrdU-labeling indices of lactotrophs in a dose- and incubation time-dependent manner. The cAMP-increasing agents were also effective in increasing BrdU-labeling indices in populations enriched for lactotrophs by differential sedimentation. The stimulatory action of forskolin was observed, regardless of concentrations of insulin that were added in combination with forskolin. Inhibition of the action of endogenous cAMP by H89 or KT5720, a protein kinase A inhibitor, attenuated an increase in BrdU-labeling indices by insulin treatment. On the other hand, the specific mitogen-activated protein kinase inhibitor PD98059, which was effective in blocking the mitogenic action of insulin, markedly suppressed the forskolin-induced increase in BrdU-labeling indices. (Bu)2cAMP antagonized not only inhibition of BrdU labeling indices but also changes in cell shape induced by bromocriptine treatment, although forskolin did not have such an antagonizing effect. These results suggest that: 1) intracellular cAMP plays a stimulatory role in the regulation of lactotroph proliferation; 2) cAMP and insulin/mitogen-activated protein kinase signalings require each other for their mitogenic actions; and 3) the antimitogenic action of bromocriptine is, at least in part, caused by inhibition of cAMP production.
Inhibition of Androgen Synthesis in Human Testicular and Prostatic Microsomes and in Male Rats by Novel Steroidal Compounds*
Authors: Nnane Ivo P. , Kato Katsuya , Liu Yang , Long Brian J. , Lu Qing , Wang Xin , Ling Yang-zhi , Brodie Angela 
Published: January 2014

AbstractThe C17,20-lyase and 5α-reductase are key enzymes in the biosynthesis of androgens. The effects of novel steroidal compounds were evaluated as inhibitors against both human C17,20-lyase and 5α-reductase in vitro. The concentrations of testosterone (T) and dihydrotestosterone (DHT) in the prostate, testis and serum and changes in the tissue weights were also determined in rats treated with the novel inhibitors. L-12 and L-26 showed potent inhibition of human testicular C17,20-lyase with IC50 values of 50 and 25 nm, respectively. L-12, L-38, and I-47 showed moderate inhibition of human testicular C17,20-lyase with IC50 values of 75, 108, and 70 nm, respectively similar to ketoconazole (78 nm). Interestingly, L-6, L-26, and L-38 also showed some inhibitory activity against 5α-reductase with IC50 values of 75, 125, and 377 nm, respectively. Finasteride, an inhibitor of 5α-reductase had an IC50 value of 33 nm. However, ketoconazole did not inhibit 5α-reductase nor did finasteride inhibit C17,20-lyase. Treatment of normal male rats with several of these novel inhibitors (50 mg/kg·day, sc, for 14 consecutive days) caused about 45–91% decrease in serum, testicular and prostatic T concentration. Similarly, serum and prostatic DHT concentration were significantly decreased in rats treated with these novel compounds by 50–90% compared with controls. Surgical castration caused almost complete elimination of circulating T and DHT concentration in rat tissues. L-6 and L-12 were the most effective and reduced the wet weight of the prostate by 50%. Although future improvements in their bioavailability are necessary, these novel steroidal compounds show promise as potential agents for reducing T and DHT levels in patients with androgen dependent diseases.
Intracellular Mechanisms Involved in Dopamine-Induced Actin Cytoskeleton Organization and Maintenance of a Round Phenotype in Cultured Rat Lactotrope Cells*
Authors: Nguyen Bao , Carbajal M. Eloísa , Vitale María L. 
Published: January 2014

AbstractThe participation of the actin cytoskeleton in the control of PRL secretion by dopamine (DA) is not yet fully understood. Recently, we demonstrated that DA induces cortical actin assembly and stabilization in anterior pituitary PRL-secreting cells (lactotropes) that can be linked to DA-induced inhibition of PRL secretion. Here we show that DA prevents cell flattening and the formation of cytoplasmic actin cables in cultured rat lactotropes. The effects of DA were reversible, mediated by D2 receptors, exclusive to lactotropes, and independent of other anterior pituitary cells present in the cultures. Because cAMP and Ca2+ mediate DA-induced inhibition of PRL secretion and synthesis, we investigated whether morphological responses to DA were dependent on these second messengers. Either inhibition of protein kinase A activity with the specific inhibitor KT5720 or blockade of Ca2+ channels with nifedipine inhibited cell flattening and induced cytoplasmic actin filament breakdown. Nifedipine was as effective as DA, but KT5720 was less effective than DA. Increased intracellular cAMP levels provoked cell flattening, which was blocked by nifedipine and KT5720, but not by DA. The results suggest that Ca2+-dependent pathways control cell shape in most lactotropes; however, in a subpopulation of lactotropes, cAMP-dependent pathways may also contribute to DA morphological responses. Next, we studied the participation of the Rho family of guanosine triphosphatases, which is known to regulate the dynamics of actin filaments. Inactivation of Rho by C3 exoenzyme induced cytoplasmic actin cable disassembly and lactotrope rounding up. No additive effects were observed among Rho-, cAMP-, and Ca2+-dependent pathways. However, C3-induced morphological responses were blocked by increased cAMP levels, suggesting that Rho-dependent steps are upstream cAMP-dependent steps. DA-induced actin cytoskeleton reorganization in lactotropes may involve modifications in the expression and localization of actin-binding proteins. DA increased expression of the actin anchoring proteins talin and α-actinin, but not of vinculin. DA enhanced association of talin to cell membranes. Increased talin-membrane interaction may be implicated in DA-induced maintenance of a round phenotype in lactotrope cells.
Enhancement of A Spermatogonial Proliferation and Differentiation in Irradiated Rats by Gonadotropin-Releasing Hormone Antagonist Administration1
Authors: Shuttlesworth Gladis A. , De Rooij Dirk G. , Huhtaniemi Ilpo , Reissmann Thomas , Russell Lonnie D. , Shetty Gunapala , Wilson Gene , Meistrich Marvin L. 
Published: January 2014

Abstract
The initial changes in the numbers, proliferation, and differentiation of A spermatogonia in irradiated rats after the administration of a GnRH antagonist, which is known to induce differentiation in this system, were investigated. LBNF1 rats were given 6 Gy ofγ -irradiation; some were treated with the GnRH antagonist Cetrorelix beginning 15 weeks after irradiation. Although the spermatogonia in the irradiated rats without hormone treatment continue to proliferate (labeling and mitotic indexes of 24% and 18%, respectively), they underwent apoptosis (apoptotic indexes of 21% by the terminal transferase-mediated end labeling assay and 9% by nuclear morphology), resulting in a constant number of A spermatogonia. Whole mount analysis of clones of A spermatogonia revealed that larger clones were more likely to undergo apoptosis than mitosis. Hormone administration decreased the intratesticular testosterone concentration to 6% of the level in irradiated rats within 1 week. Concomitantly, there was a decrease in spermatogonial apoptotic indexes to 43% of levels in irradiated-only rats, leading to an increases in their numbers by 150%, their diameters by 11%, and their labeling indexes by 31%. The sizes of the mitotic clones gradually increased, and clones of more than eight cells appeared at week 3 of hormone treatment. A spermatogonial differentiation began at week 4, and by week 6.6, differentiation occurred in 30% of the tubules. Thus, suppression of intratesticular testosterone by the GnRH antagonist may be responsible for the immediate changes in spermatogonial numbers and kinetics, but several additional steps are required before differentiation begins, which did not occur until week 4.
Whole-Cell Recordings from Preoptic/Hypothalamic Slices Reveal Burst Firing in Gonadotropin-Releasing Hormone Neurons Identified with Green Fluorescent Protein in Transgenic Mice*
Authors: Suter Kelly J. , Wuarin Jean-Pierre , Smith Bret N. , Dudek F. Edward , Moenter Suzanne M. 
Published: January 2014

Abstract
Central control of reproduction is governed by a neuronal pulse generator that underlies the activity of hypothalamic neuroendocrine cells that secrete GnRH. Bursts and prolonged episodes of repetitive action potentials have been associated with hormone secretion in this and other neuroendocrine systems. To begin to investigate the cellular mechanisms responsible for the GnRH pulse generator, we used transgenic mice in which green fluorescent protein was genetically targeted to GnRH neurons. Whole-cell recordings were obtained from 21 GnRH neurons, visually identified in 200-μm preoptic/hypothalamic slices, to determine whether they exhibit high frequency bursts of action potentials and are electrically coupled at or near the somata. All GnRH neurons fired spontaneous action potentials, and in 15 of 21 GnRH neurons, the action potentials occurred in single bursts or episodes of repetitive bursts of high frequency spikes (9.77 ± 0.87 Hz) lasting 3–120 sec. Extended periods of quiescence of up to 30 min preceded and followed these periods of repetitive firing. Examination of 92 GnRH neurons (including 32 neurons that were located near another green fluorescent protein-positive neuron) revealed evidence for coupling in only 1 pair of GnRH neurons. The evidence for minimal coupling between these neuroendocrine cells suggests that direct soma to soma transfer of information, through either cytoplasmic bridges or gap junctions, has a minor role in synchronization of GnRH neurons. The pattern of electrical activity observed in single GnRH neurons within slices is temporally consistent with observations of GnRH release and multiple unit electrophysiological correlates of LH release. Episodes of burst firing of individual GnRH neurons may represent a component of the GnRH pulse generator.
Diametric Effects of Bacterial Endotoxin Lipopolysaccharide on Adrenal and Leydig Cell Steroidogenic Acute Regulatory Protein
Authors: Held Hales Karen , Diemer Thorsten , Ginde Salil , Shankar Birinder K. , Roberts Maretha , Bosmann H. Bruce , Hales Dale B. 
Published: January 2014

AbstractImmune activation results in the activation of adrenal steroidogenesis and inhibition of gonadal steroidogenesis. Previous studies indicated that these effects were caused primarily by activation and suppression of the secretion of ACTH and LH, respectively. However, other evidence indicated a direct effect of the immune system on the gonads. In this study, serum testosterone, quantitated by RIA after lipopolysaccharide injection, showed a significant decrease within 2 h. Parallel measurement of serum LH showed no change. There were no differences in LH receptor or cAMP produced in Leydig cells between vehicle- and lipopolysaccharide-injected mice. The 30-kDa form of the steroidogenic acute regulatory (StAR) protein was quantitated, by Western blot, in Leydig cells and was found to decrease in a time-dependent manner. No change in StAR protein messenger RNA (mRNA) was detected by Northern analysis during this time, nor were any changes found in the levels of mRNA for the steroidogenic enzymes P450scc, 3β-hydroxysteroid dehydrogenaseΔ 4-Δ5-isomerase, or P450c17. In the adrenal, StAR protein was increased, as was StAR protein mRNA. No changes were observed in the levels of mRNA for P450scc, 3β-hydroxysteroid dehydrogenaseΔ 4-Δ5-isomerase, or P450c21. Thus, although the mechanisms of regulation differ, changes in the levels of StAR protein are a sensitive indicator of the steroidogenic capacity of these two tissues.
Regulation of Thyroid Follicular Cell Function by Intracellular Redox-Active Copper*
Authors: Iseki Atsushi , Kambe Fukushi , Okumura Kenji , Hayakawa Tetsuo , Seo Hisao 
Published: January 2014

AbstractPyrrolidine dithiocarbamate (PDTC) is a metal-chelating compound that exerts prooxidant or antioxidant effects and is widely used to study redox regulation of cell function. In the present study, we investigated effects of PDTC on the function of rat thyroid follicular FRTL-5 cells. Treatment of the cells with PDTC resulted in a marked decrease in Pax-8 messenger RNA level and its DNA-binding activity. This decrease was associated with a significant reduction in thyroperoxidase (TPO) messenger RNA level. Expression of TTF-1 and thyroglobulin was not affected by PDTC. Treatment with PDTC also decreased DNA-binding activity of p53, a tumor suppressor protein, and increased cell proliferation rates. These changes were not observed by the treatment with another antioxidant, N-acetyl-lcysteine, suggesting that the metal-chelating, prooxidant property of PDTC is responsible for its effects. Indeed, the intracellular level of copper was significantly increased by PDTC. Treatment with bathocuproinedisulfonic acid, a noncell-permeable chelator of Cu1+, abrogated the copper increase by PDTC and its effects on Pax-8 and TPO expression as well as on p53 binding. Taken together, these results indicate that the intracellular level of redox-active copper is crucial for Pax-8 and TPO expression and for proliferation of thyroid follicular cells.