bluuebunny/crossref_metadata_2025_split huggingface dataset:
Existence of Galanin in Lumbosacral Sympathetic Ganglionic Neurons That Project to the Quail Uterine Oviduct*
Authors: Sakamoto Hirotaka , Ubuka Takayoshi , Kohchi Chie , Li Dan , Ukena Kazuyoshi , Tsutsui Kazuyoshi 
Published: January 2014

Abstract
Oviposition in birds is conducted by vigorous contractions of the uterine oviduct. We recently isolated an oviposition-inducing peptide that was identified as avian galanin from mature quail oviducts. This peptide was localized in neuronal fibers terminating in muscle layers in the uterine oviduct and evoked vigorous uterine contractions through binding to receptors located in the uterus. However, no cell bodies that express avian galanin were detected in the uterus or other oviduct regions. To understand the control mechanism of avian oviposition by galanin, we identified the neurons that synthesize galanin and project to the uterus with the combination of retrograde labeling with neurobiotin and immunocytochemistry for galanin in mature Japanese quails. Retrograde labeling with neurobiotin from the uterus revealed that lumbosacral sympathetic ganglionic neurons located in the uterine side projected their axons to the uterine muscle layer. Abundant elementary granules were observed in somata of the retrogradely labeled sympathetic ganglionic neurons, suggesting that labeled neurons may function as a neurosecretory cell. Immunocytochemical analysis with the antiserum against avian galanin showed an intense immunoreaction restricted to somata of the retrograde-labeled ganglionic neurons. Preabsorbing the antiserum with avian galanin resulted in a complete absence of the immunoreaction. Competitive enzyme-linked immunosorbent assay using antigalanin serum confirmed that avian galanin existed in the sympathetic ganglionic neurons. Expression of the avian galanin messenger RNA in the neurons was further verified by Northern blot analysis. In addition, both avian galanin and its messenger RNA in the neurons were highly expressed in mature birds, unlike in immature birds.
These results suggest that lumbosacral sympathetic ganglionic neurons innervating the uterine muscle produce avian galanin in mature birds. Because this peptide acts directly on the uterus to evoke oviposition through a mechanism of the induction of vigorous uterine contraction, galaninergic innervation of the uterine oviduct may be essential for avian oviposition.
Adrenergic Regulation of Mitogen-Activated Protein Kinase in Rat Pinealocytes: Opposing Effects of Protein Kinase A and Protein Kinase G**This work was supported by grants from the Medical Research Council of Canada.
Authors: Ho A. K. , Chik C. L. 
Published: January 2014

AbstractThe role of adrenergic stimulation in the regulation of mitogen-activated protein kinase (MAPK) in rat pinealocytes was investigated by measuring phosphorylated MAPK using Western blot analysis and a MAPK enzymatic assay. Stimulation with the endogenous neurotransmitter, norepinephrine (NE; a mixed α- and β-adrenergic agonist), concentration dependently increased the phosphorylation of both p44 and p42 isoforms of MAPK. This effect of NE was blocked by PD98059 and UO126 (two inhibitors of MEK). Treatment with prazosin or propranolol significantly reduced the effect of NE on MAPK phosphorylation, suggesting the involvement of both α- andβ -adrenergic receptors. Investigation into the intracellular mechanisms of NE action revealed that the increase in MAPK phosphorylation was blocked by KT5823 (a protein kinase G inhibitor), but was enhanced by H89 (a protein kinase A inhibitor). Calphostin C (a protein kinase C inhibitor) and KN93 (a Ca2+/calmodulin-dependent protein kinase inhibitor) also attenuated NE-mediated MAPK activation, but to a lesser degree. Furthermore, inhibition of MAPK phosphorylation by (Bu)2cAMP was effective in reducing MAPK activation by (Bu)2cGMP, an active phorbol ester or ionomycin. These results indicate that the effect of NE on MAPK phosphorylation represents mainly the integration of two signaling mechanisms, protein kinase A and protein kinase G, each having an opposite effect on MAPK phosphorylation.
Matrix γ-Carboxyglutamic Acid Protein Is a Key Regulator of PTH-Mediated Inhibition of Mineralization in MC3T3-E1 Osteoblast-Like Cells
Authors: Gopalakrishnan Rajaram , Ouyang Hongjiao , Somerman Martha J. , McCauley Laurie K. , Franceschi Renny T. 
Published: January 2014

AbstractAs part of its overall function as a major regulator of calcium homeostasis, PTH stimulates bone resorption and inhibits osteoblast-mediated biomineralization. To determine the basis for the inhibitory actions of this hormone, we compared the time course of PTH-dependent inhibition of mineralization in MC3T3-E1 osteoblast-like cells with changes in mRNA levels for several extracellular matrix proteins previously associated either with induction or inhibition of mineralization. Mineralizing activity was rapidly lost in PTH-treated cells (∼30% inhibition after 3 h, 50% inhibition at 6 h). Of the proteins examined, changes in matrix γ-carboxyglutamic acid protein were best correlated with PTH-dependent inhibition of mineralization. Matrix γ-carboxyglutamic acid protein mRNA was rapidly induced 3 h after PTH treatment, with a 6- to 8-fold induction seen after 6 h. Local in vivo injection of PTH over the calvaria of mice also induced a 2-fold increase in matrix γ-carboxyglutamic acid protein mRNA. Warfarin, an inhibitor of matrix γ-carboxyglutamic acid protein γ-carboxylation, reversed the effects of PTH on mineralization in MC3T3-E1 cells, whereas vitamin K enhanced PTH activity, as would be expected if a γ-carboxyglutamic acid-containing protein were required for PTH activity. Levels of the other mRNAs examined were not well correlated with the observed changes in mineralization. Osteopontin, an in vitro inhibitor of mineralization, was induced approximately 4-fold 12 h after PTH addition. Bone sialoprotein mRNA, which encodes an extracellular matrix component most frequently associated with mineral induction, was inhibited by 50% after 12 h of PTH treatment. Osteocalcin mRNA, encoding the other known γ-carboxyglutamic acid protein in bone, was also inhibited by PTH, but, again, with a significantly slower time course than was seen for mineral inhibition. Taken together, these results show that the rapid inhibition of osteoblast mineralization induced by in vitro PTH treatment is at least in part explained by induction of matrix γ-carboxyglutamic acid protein.
Cyclical Regulation of GnRH Gene Expression in GT1–7 GnRH-Secreting Neurons by Melatonin
Authors: Roy Deboleena , Angelini Nadia L. , Fujieda Hiroki , Brown Gregory M. , Belsham Denise D. 
Published: January 2014

AbstractThe pineal hormone melatonin plays an important role in the neuroendocrine control of reproductive physiology, but its effects on hypothalamic GnRH neurons are not yet known. We have found that GT1–7 GnRH-secreting neurons express membrane-bound G protein-coupled melatonin receptors, mt1 (Mel-1a) and MT2 (Mel-1b) as well as the orphan nuclear receptors RORα and RZRβ. Melatonin (1 nm) significantly downregulates GnRH mRNA levels in a 24-h cyclical manner, an effect that is specifically inhibited by the melatonin receptor antagonist luzindole (10 μm). Repression of GnRH gene expression by melatonin appears to occur at the transcriptional level and can be mapped to the GnRH neuron-specific enhancer located within the 5′ regulatory region of the GnRH gene. Using transient transfection of GT1–7 cells, downregulation of GnRH gene expression by melatonin was further localized to five specific regions within the GnRH enhancer including −1827/−1819,− 1780/−1772, −1746/−1738, −1736/−1728, and −1697/−1689. Interestingly, the region located at −1736/−1728 includes sequences that correspond to two direct repeats of hexameric consensus binding sites for members of the ROR/RZR orphan nuclear receptor family. To begin to dissect the mechanisms involved in the 24-h cyclical regulation of GnRH transcription, we have found that melatonin (10 nm) induces rapid internalization of membrane-bound mt1 receptors through a β-arrestin 1-mediated mechanism. These results provide the first evidence that melatonin may mediate its neuroendocrine control on reproductive physiology through direct actions on the GnRH neurons of the hypothalamus, both at the level of GnRH gene expression and through the regulation of G protein-coupled melatonin receptors.
Increase in β-Cell Mass in Transplanted Porcine Neonatal Pancreatic Cell Clusters Is Due to Proliferation of β-Cells and Differentiation of Duct Cells*
Authors: Trivedi N. , Hollister-Lock J. , Lopez-Avalos M. D. , O’Neil J. J. , Keegan M. , Bonner-Weir S. , Weir G. C. 
Published: January 2014

Abstract
A 20-fold increase in β-cell mass has been found after transplantation of porcine neonatal pancreatic cell clusters (NPCCs). Here the mechanisms leading to this increased β-cell mass were studied. NPCCs (4000 islet equivalents) generated after 8 days culture of digested neonatal pig pancreas were transplanted beneath the renal capsule of streptozotocin (STZ) diabetic and normoglycemic nude mice. Grafts were removed at 10 days, 6 weeks, and 20 weeks after transplantation for immunostaining and insulin content. Proliferation of β-cells and duct cells was assessed morphometrically using double immunostaining for Ki-67 with insulin or cytokeratin 7 (CK7). Graft maturation was assessed with double immunostaining of CK7 and insulin. Apoptosis was determined using propidium iodide staining. β-cell proliferation in NPCCs was higher after 8 days of culture compared with that found in neonatal pig pancreas. After transplantation, β-cell proliferation remained high at 10 days, decreased somewhat at 6 weeks, and was much lower 20 weeks after transplantation. Diabetic recipients not cured at 6 weeks after transplantation had significantly higherβ -cell proliferation compared with those cured and to normoglycemic recipients. The size of individual β-cells, as determined by cross-sectional area, increased as the grafts matured. Graft insulin content was 20-fold increased at 20 weeks after transplantation compared with 8 days cultured NPCCs. The proliferation index of duct cells was significantly higher in neonatal pig pancreas than in 8 days cultured NPCCs and in 10-day-old grafts. The incidence of apoptosis in duct cells appeared to be low. About 20% of duct cells 10 days post transplantation showed costaining for CK7 and insulin, a marker of protodifferentiation. In conclusion, the increase in β-cell mass after transplantation of NPCCs is due to both proliferation of differentiated β-cells and differentiation of duct cells intoβ -cells.
Gonadotropins Decrease Estrogen Receptor-β Messenger Ribonucleic Acid Stability in Rat Granulosa Cells*
Authors: Guo Caixia , Savage Lisa , Sarge Kevin D. , Park-Sarge Ok-Kyong 
Published: January 2014

AbstractWe have previously shown that the preovulatory LH surge down-regulates estrogen receptor-β (ERβ) messenger RNA (mRNA) levels selectively in the granulosa cells of preovulatory follicles. To gain insight into the underlying mechanisms, we examined whether the LH-induced loss of ERβ mRNA expression in rat granulosa cells is attributable to the hormone-induced changes at the level of transcription and/or mRNA degradation. When the rate of ERβ gene transcription was assessed in cultured granulosa cells, by nuclear run-off assays, we observed only a marginal effect of hCG on ERβ gene transcription. In contrast, when ERβ mRNA levels were estimated in granulosa cells that were cultured in the presence of 5,6-dichloro-1-β-d-ribofuranosylbenzimidazole (DRB), an RNA synthesis inhibitor, we observed a significant inhibitory effect of human CG (hCG) on ERβ mRNA expression at a magnitude similar to that observed in the absence of DRB. Forskolin (FSK) and 2-O-tetradecanol-phorbol-13-acetate (TPA), pharmacological agents that mimic LH actions in granulosa cells, also showed similar effects. Thus, these results suggest that LH decreases ERβ mRNA expression in the granulosa cells of preovulatory follicles, primarily by destabilizing the preexisting ERβ mRNA. We next determined the decay rate of the ERβ mRNA in granulosa cells that were cultured in the presence of DRB and additional hCG, FSK, or TPA for various time periods, by estimating ERβ mRNA levels, using semiquantitative RT-PCR assays and subsequent linear regression analyses. The half-life of the ERβ mRNA in the presence of vehicle was 17.87 ± 1.2 h (n = 4). hCG dramatically decreased the half-life of the ERβ mRNA (4.85 ± 0.49 h, n = 4). Similarly, both FSK and TPA decreased the half-life of the ERβ mRNA to 3.57 ± 0.31 h and 4.02± 0.13 h, respectively. We extended these findings by examining whether the LH-induced down-regulation of the ERβ mRNA is cycloheximide-sensitive. When granulosa cells were cultured in the presence of cycloheximide, a protein synthesis inhibitor, the inhibitory effects of hCG, FSK, and TPA on ERβ mRNA levels were abolished. Similar results were obtained in the presence or absence of DRB, indicating that the hormone-induced destabilization of the ERβ mRNA is coupled with translation processes. Taken together, our results demonstrate that LH decreases ERβ mRNA expression, predominantly at the posttranscriptional level, in a cycloheximide-sensitive manner.
Cyr61, a Member of the CCN Family, Is Required for MCF-7 Cell Proliferation: Regulation by 17β-Estradiol and Overexpression in Human Breast Cancer
Authors: Sampath Deepak , Winneker Richard C. , Zhang Zhiming 
Published: January 2014

Abstract
Cyr61, a member of the CCN (CTGF/Cyr61/NOV) family of growth regulators, is a secreted cysteine-rich proangiogenic factor that has been implicated in tumorigenesis. Previous studies have also demonstrated that Cyr61 is regulated by 17β-estradiol (E2) in the uterus. Therefore, we hypothesized that hormonal regulation of Cyr61 may be important in estrogen-dependent pathogenic processes such as breast tumorigenesis. Our study demonstrates that both Cyr61 messenger RNA and protein are induced by E2 in MCF-7 mammary adenocarcinoma cells that primarily overexpress estrogen receptor α (ERα) in a dose-dependent and immediate early fashion. Cyr61 gene induction by E2 is transcriptionally regulated by ERα as the antiestrogen, ICI 182,780, and actinomycin D blocked induction completely. In addition, Cyr61 is up-regulated in MCF-7 cells by epidermal growth factor (EGF) in an immediate early fashion as well. The functional relevance of steroid induction of Cyr61 in breast cancer cell growth is demonstrated by anti-Cyr61 neutralizing antibodies, which diminished E2 and EGF-dependent DNA synthesis and dramatically reduced E2-driven cell proliferation by more than 70%. Most importantly, Cyr61 is overexpressed in 70% (28 of 40) of breast cancer patients with infiltrating ductal carcinoma and is localized exclusively to hyperplastic ductal epithelial cells. Moreover, the levels of Cyr61 protein are higher in breast tumors that are ER+/EGF receptor+ than those that are ER−/EGF receptor+, suggesting that estrogens may mediate Cyr61 expression in vivo. Collectively, our data suggest that Cyr61 may play a critical role in estrogen- as well as growth factor-dependent breast tumor growth.
Disease Progression in Chronic Relapsing Experimental Allergic Encephalomyelitis Is Associated with Reduced Inflammation-Driven Production of Corticosterone
Authors: Stefferl Andreas , Storch Maria K. , Linington Christopher , Stadelmann Christine , Lassmann Hans , Pohl Thomas , Holsboer Florian , Tilders Fred J. H. , Reul Johannes M. H. M. 
Published: January 2014

AbstractIn this study, we demonstrate that disruption of neuroendocrine signaling is a major factor driving disease progression in myelin oligodendrocyte glycoprotein-induced chronic relapsing experimental autoimmune encephalomyelitis, an animal model of multiple sclerosis. Although the initial episode of chronic relapsing experimental autoimmune encephalomyelitis is associated with a robust hypothalamic-pituitary-adrenocortical axis response, we show that subsequent disease progression is associated with a selective desensitization of hypothalamic-pituitary-adrenocortical responsiveness to inflammatory mediators. Inflammatory activity in the central nervous system during relapse is therefore unable to produce an endogenous immunosuppressive corticosterone response, and disease progresses into an ultimately lethal phase. However, disease progression is inhibited if the circulating corticosterone level is maintained at levels seen during the initial phase of disease. The effect of hypothalamic-pituitary-adrenocortical axis desensitization on the clinical course of experimental autoimmune encephalomyelitis is aggravated by a marked reduction in proinflammatory cytokine synthesis in the central nervous system in the later stages of disease, reflecting an increasing involvement of antibody, rather than T cell-dependent effector mechanisms, in disease pathogenesis, with time. Thus, our data indicate that distinct immune-endocrine effects play a decisive role in determining disease progression in multiple sclerosis, a concept supported by reports that a subpopulation of multiple sclerosis patients shows evidence of hypothalamic-pituitary-adrenocortical axis desensitization.
Expression of 11β-Hydroxylase in Rat Leydig Cells
Authors: Wang Gui-Min , Ge Ren-Shan , Latif Syed A. , Morris David J. , Hardy Matthew P. 
Published: January 2014

Abstract11β-Hydroxy (11β-OH) derivatives of certain steroids function as inhibitors of 11β-hydroxysteroid dehydrogenase isoform 1 (11βHSD1), an enzyme expressed in Leydig cells that catalyzes the reversible oxidation of biologically active glucocorticoids to inactive 11-dehydro metabolites. 11β-Hydroxylase is an adrenal enzyme responsible for glucocorticoid biosynthesis, catalyzing 11β-hydroxylation of steroids and thus producing 11β-OH-steroid derivatives. The aims of the present study were 1) to examine whether 11β-hydroxylase is expressed in testis, 2) to define the biochemical characteristics of the testicular form of this enzyme, and 3) to establish whether 11β-hydroxylated steroids inhibit Leydig cell 11βHSD1 activities. 11β-Hydroxylase mRNA was detected in purified rat Leydig cells by RT-PCR. Sequencing confirmed that the PCR products had 100% identity with the published rat adrenal enzyme cDNA sequence. Immunohistochemistry and Western blot analysis using a mouse monoclonal antibody confirmed the expression of 11β-hydroxylase protein in Leydig cells. Moreover, 11β-hydroxylase activity, synthesis of corticosterone from 11-deoxycorticosterone, was measurable in Leydig cells, and the Km and maximum velocity values were 7.28 ± 0. 92 μm and 1.13 ± 0.04 μmol/106 cell·h, respectively. When assayed in Leydig cells, several 11β-hydroxylated steroids were efficient inhibitors of 11βHSD1 dehydrogenase activity, whereas other 11-keto compounds were effective as inhibitors of oxidoreductase activity. These results provide the first direct evidence that rat Leydig cells express 11β-hydroxylase, which may be involved in the regulation of glucocorticoid metabolism within the testis through local biosynthesis of endogenous inhibitors of 11βHSD1.
TNFα Potently Activates Osteoclasts, through a Direct Action Independent of and Strongly Synergistic with RANKL
Authors: Fuller Karen , Murphy Chiho , Kirstein Barrie , Fox Simon W. , Chambers Timothy J. 
Published: January 2014

AbstractTNFα is pivotal to the pathogenesis of inflammatory and possibly postmenopausal osteolysis. Much recent work has clarified mechanisms by which TNFα promotes osteoclastogenesis, but the means by which it activates osteoclasts to resorb bone remain uncertain. We found that very low concentrations of TNFα promoted actin ring formation, which correlates with functional activation in osteoclasts, both in osteoclasts formed in vitro and extracted from newborn rats. TNFα was equipotent with RANKL for this action. Activation by TNFα was unaffected by blockade of RANKL by OPG, its soluble decoy receptor, suggesting that this was due to a direct action on osteoclasts. Bone resorption was similarly directly and potently stimulated, in a RANKL-independent manner in osteoclasts, whether these were formed in vitro or in vivo. Interestingly, TNFα promoted actin ring formation at concentrations an order of magnitude below those required for osteoclastic differentiation. Moreover, TNFα strongly synergized with RANKL, such that miniscule concentrations of TNFα were sufficient to substantially augment osteoclast activation. The extreme sensitivity of osteoclasts to activation by TNFα suggests that the most sensitive osteolytic response of bone to TNFα is through activation of existing osteoclasts; and the strong synergy with RANKL provides a mechanism whereby increased osteolysis can be achieved without disturbance to the underlying pattern of osteoclastic localization.
Effect of GABA on GnRH Neurons Switches from Depolarization to Hyperpolarization at Puberty in the Female Mouse
Authors: Han Seong-Kyu , Abraham Istvan M. , Herbison Allan E. 
Published: January 2014

Abstract
The amino acid γ-aminobutyric acid (GABA) plays an important role in the regulation of the GnRH neurons. We examined whether GABA depolarizes or hyperpolarizes GnRH neurons over postnatal development using gramicidin, perforated-patch electrophysiology combined with GnRH-LacZ transgenic mice in whom GnRH neurons can be made to fluoresce. The basic membrane properties and GABA responsiveness of GnRH neurons were not altered by transgene expression or fluorescence. Ten of 12 immature GnRH neurons (10–17 d) were depolarized by GABA in a direct and dose-dependent manner that was blocked by a GABAA receptor antagonist. In peripubertal GnRH neurons (25–30 d), GABA exerted depolarizing (4/11) as well as hyperpolarizing (5/11) effects on GnRH neurons. In adult female mice, GABA was found to exert exclusively hyperpolarizing actions on GnRH neurons (9/10) that were direct and mediated by the GABAA receptor. GABA switched from depolarizing to hyperpolarizing actions around postnatal d 31, the time of vaginal opening. Unidentified preoptic area neurons exhibited predominantly hyperpolarizing responses to GABA at all three postnatal stages. These findings demonstrate that GnRH neurons display an unusually late postnatal switch in their response to GABA. They also provide the first direct evidence that GABA inhibits the electrical activity of postpubertal GnRH neurons.