bluuebunny/crossref_metadata_2025_split huggingface dataset:
Expression of p27Kip1 in Osteoblast-Like Cells during Differentiation with Parathyroid Hormone*
Authors: Onishi Takehisa , Hruska Keith 
Published: January 2014

AbstractPTH is a major systemic regulator of bone metabolism and plays an important role in both bone formation and resorption. PTH either inhibits or stimulates osteoblastic cell proliferation depending on the model that is studied. We analyzed the cell cycle of the UMR-106 cell line, a relatively differentiated osteoblastic osteogenic sarcoma line in which PTH is known to inhibit proliferation but the mechanism of action is unknown. PTH decreased the proportion of cells in S phase and increased the number of G1 phase cells. We examined the effect of PTH on the regulators of the G1 phase cyclin-dependent kinases and found that PTH increased p27Kip1, but not p21Cip1, levels. This effect was mimicked by 8-bromo-cAMP, but not by phorbol 12-myristate 13-acetate. The protein kinase A inhibitor KT5720 abolished the effect of PTH on the increase in p27Kip1 expression. PTH increased CDK2-associated p27Kip1 without affecting the levels of CDK2. CDK2 activity was down-regulated by both PTH and 8-bromo-cAMP treatment. These data suggest that PTH blocks entry of cells into S phase and inhibits cell proliferation as the consequence of an increase in p27Kip1, which is mediated through the protein kinase A pathway. The inhibition of G1 cyclin-dependent kinases by p27Kip1 could cause a reduction of phosphorylation of key substrates and inactivation of transcription factors essential for entry into S phase. The inhibition of cell cycle progression through PKA-mediated p27Kip1 induction might play an important role in PTH-induced differentiation of osteoblasts.
Orthovanadate Stimulates Cyclic Guanosine Monophosphate-Inhibited Cyclic Adenosine Monophosphate Phosphodiesterase Activity in Isolated Rat Fat Pads through Activation of Particulate Myelin Basic Protein Kinase by Protein Tyrosine Kinase
Authors: Ueki Hiroshi , Mitsugi Shuichi , Kawashima Yoshihito , Motoyashiki Toshio , Morita Tetsuo 
Published: January 2014

AbstractInvolvement of protein kinases in the stimulation of cGMP-inhibited cAMP phosphodiesterase (PDE) activity by orthovanadate (vanadate) was studied. When the fat pads were incubated with 2 mm vanadate or 10 nm insulin, the stimulation of myelin basic protein kinase (MBPK) activity in the particulate by vanadate reached a maximum at 60 min. In contrast, insulin showed a transient increase at 20 min. A 60-min incubation of the fat pads with vanadate stimulated all activities of protein tyrosine kinase (PTK), MBPK, and PDE in the particulate, in a similar dose-dependent manner. Amiloride, a PTK inhibitor, inhibited the stimulations of three enzymes by vanadate in a similar concentration range. Enzyme fractions, which were separated from the solubilized particulate, were subjected to the immunoblot analysis. A fraction of MBPK was identified to contain a major protein of mol wt (44K) and a minor one (42K), both of which are immunoreactive with a mitogen-activated protein kinase (MAPK) antibody. The partially purified PDE activity was stimulated by the addition of the partially purified MBPK. The further stimulation was observed with the PTK-activated MBPK. These results suggest that vanadate stimulates in part the PDE activity through the activation of the particulate MBPK, probably MAPKs, by PTK sensitive to vanadate.
The Formation of Thyrotropin Receptor (TSHR) Antibodies in a Graves’ Animal Model Requires the N-Terminal Segment of the TSHR Extracellular Domain
Authors: Kikuoka Shuichi , Shimojo Naoki , Yamaguchi Ken-Ichi , Watanabe Yukihiko , Hoshioka Akira , Hirai Aizan , Saito Yasushi , Tahara Kazuo , Kohn Leonard D. , Maruyama Naoki , Kohno Yoichi , Niimi Hiroo 
Published: January 2014

Abstract
Immunization of AKR/N mice with murine fibroblasts, transfected with the TSH receptor (TSHR) and a murine major histocompatibility complex class II molecule having the same H-2k haplotype (but not either alone), induces immune thyroid disease with the humoral and histological features of human Graves’, including the presence of two different TSHR antibodies (TSHRAbs): stimulating TSHRAbs, which cause hyperthyroidism; and TSH-binding-inhibiting immunoglobulins. The primary functional epitope for both types of antibodies in Graves’ patients is on the N-terminal portion of the extracellular domain of the TSHR, residues 25 to 165; most require residues 90–165 to express TSHRAb activity, as evidenced in studies using chimeras of the TSHR and lutropin-choriogonadotropin receptor (LH-CGR). To evaluate the role of this region of the TSHR in the formation of Graves’ TSHRAbs, we immunized AKR/N mice with fibroblasts transfected with three human TSHR chimeras with residues 9–165 (Mc1+2), 90–165 (Mc2), or 261–370 (Mc4) substituted by equivalent residues of the rat LH-CGR. Mice immunized with the Mc1+2 and Mc2 chimeras, with the N-terminal portion of the extracellular domain of the TSHR substituted by LH-CGR residues, did not develop TSHRAbs. Mice immunized with the Mc4 chimera, having a major portion of the C-terminal portion of the extracellular domain of the TSHR replaced by comparable LH-CGR residues, can develop TSHRAbs. The results suggest that the N-terminal segment of the TSHR extracellular domain is not only a critical functional epitope for Graves’ TSHRAbs, but it is important also in their formation in a mouse model of Graves’ disease.
5-Lipoxygenase Metabolites Inhibit Bone Formation in Vitro
Authors: Traianedes Kathy , Dallas Mark R. , Garrett I. Ross , Mundy Gregory R. , Bonewald Lynda F. 
Published: January 2014

AbstractThe leukotrienes and peptido-leukotrienes are 5-lipoxygenase (5-LO) metabolites of arachidonic acid that appear to have unique effects on bone, distinct from those of the prostaglandins. Application of exogenous leukotrienes in vitro and in vivo results in increased osteoclast formation and bone resorption. While 5-LO metabolites of arachidonic acid clearly stimulate osteoclastic bone resorption, little is known concerning their effects on osteoblastic bone formation. We examined the effects of the 5-LO metabolites 5-HETE, the leukotriene LTB4 and, as representative of the peptido-leukotrienes, LTD4 on the formation of mineralized nodules of fetal rat calvarial cells in the presence of dexamethasone and recombinant human bone morphogenetic protein-2 (rhBMP-2). We also examined the effects of these 5-LO metabolites on alkaline phosphatase activity and cell proliferation in these cultures and the effects of 5-HETE and LTB4 on cultured explants of neonatal murine calvariae. We found that the bone-forming capacity of osteoblasts was impaired when cells were cultured in the presence of 5-LO metabolites. These data indicate that metabolites of the 5-LO pathway are negative regulators of bone formation. The continued presence of these metabolites in the bone environment might account, in part, for the bone loss associated with chronic inflammatory conditions.
Effects of Growth Hormone Secretagogues on Prolactin Release in Anesthetized Dwarf (dw/dw) Rats*
Authors: Carmignac Danielle F. , Bennett Pamela A. , Robinson Iain C. A. F. 
Published: January 2014

AbstractIn addition to stimulating GH release, GH secretagogues such as GH-releasing peptide-6 (GHRP-6) stimulate small amounts of ACTH and PRL release. Although the effects on ACTH have recently been studied, there is little information about the effects of GHRP-6 on PRL. We have now studied GHRP-6-induced GH and PRL release and their regulation by estrogen (E2) in anesthetized male and female rats and in GH-deficient dwarf (dw/dw) rats that maintain high pituitary PRL stores and show elevated hypothalamic GH secretagogue receptor expression. Whereas GHRP-6 (0.1–2.5 μg, iv) did not induce PRL release in normal male or female rats, significant PRL responses were observed in dw/dw females. These responses were abolished by ovariectomy and could be strongly induced in male dw/dw rats by E2 treatment. These effects could be dissociated from GHRP-6-induced GH release in the same animals, but not from PRL release induced by TRH, which was also abolished by ovariectomy and induced in males by E2 treatment. However, the effects of GHRP-6 on PRL were unlikely to be mediated by TRH because in the same animals, TSH levels were unaffected by GHRP-6 whereas they were increased by TRH. The increased PRL response could reflect an increase in GH secretagogue receptor expression that was observed in the arcuate and ventromedial nuclei of E2-treated rats. Our results suggest that the minimal PRL-releasing activity of GHRP-6 in normal rats becomes prominent in GH-deficient female dw/dw rats and is probably exerted directly at the pituitary; these GHRP-6 actions may be modulated by E2 at both hypothalamic and pituitary sites.
Cyclical Alternative Exon Splicing of Transcription Factor Cyclic Adenosine Monophosphate Response Element-Binding Protein (CREB) Messenger Ribonucleic Acid during Rat Spermatogenesis*
Authors: Daniel Philip B. , Habener Joel F. 
Published: January 2014

Abstract
During spermatogenesis, the levels of cAMP in seminiferous tubules undergo stage-dependent cyclical fluctuations. We show that changes in cAMP levels are accompanied by alternative exon splicing of the RNA encoding the cAMP-responsive transcription factor CREB (cAMP response element-binding protein), expressed in both the Sertoli and germ cells. Exons Y and W are expressed exclusively in the testis, and they introduce stop codons into the normal protein coding frame of CREB. The splicing in of W was shown earlier to activate the internal translation of two alternative products of the CREB messenger RNA (mRNA) containing the DNA-binding domain (I-CREBs). The I-CREBs act as potent inhibitors of activator isoforms of CREB. The functions of the alternatively spliced exon Y are unknown. To investigate whether the splicing of exons W and Y is regulated during spermatogenesis, seminiferous tubules, isolated from adult rats, were dissected into segments representing different stages of the spermatogenic cycle and were analyzed by RT-PCR. The analyses of pooled-tubule segments revealed stage-dependent splicing of both exons W and Y in the CREB transcripts. Single tubules were dissected into smaller segments for greater staging accuracy and were analyzed by RT-PCR for CREB mRNAs containing either exons W or Y, as well as for FSH receptor mRNA. This analysis confirmed that a marked, cycle-dependent variation in CREB mRNA levels was occurring. Maximal splicing of exons W and Y occurs independently at different stages of the spermatogenic cycle, stages II-VI and IX, respectively. The distinct spermatogenic cycle-dependent regulation of the splicing of exons W and Y provides further evidence in support of a functional relevance for CREB-W and Y mRNA isoforms in spermatogenesis.
Nitric Oxide Plays an Important Role in the Diurnal Change of Tuberoinfundibular Dopaminergic Neuronal Activity and Prolactin Secretion in Ovariectomized, Estrogen/Progesterone-Treated Rats**This study was supported, in part, by Grants NSC86–2314-B010-M10 and NSC87–2314-B010–016 (to J.-T.P) from the National Science Council of the Republic of China.
Authors: YEN Shih-Hui , Pan Jenn-Tser 
Published: January 2014

Abstract
A significant diurnal change of tuberoinfundibular dopaminergic (TIDA) neuronal activity coincident with the estrogen (E2)-induced afternoon PRL surge has been reported in ovariectomized, E2-primed (OVX+E2) rats. Systemic injection of a nitric oxide (NO) synthase (NOS) inhibitor, NG-nitro-l-arginine (l-NA, 50 mg/kg, ip at 1000 and 1200 h), significantly blocked the diurnal changes of TIDA neuronal activity and PRL secretion at 1500 and 1700 h in OVX+E2 rats. Coadministration of l-arginine (300 mg/kg, ip) with l-NA completely prevented the effects of l-NA. Total nitrite/nitrate levels in the serum of l-NA- and l-NA+l-arginine-treated rats substantiated the effects of l-NA and l-arginine on NO production. Pretreatment of antisense oligodeoxynucleotide (ODN; 1μ g/3 μl; intracerebroventricularly at 48, 24, and 7 h before sacrifice) against the messenger RNA (mRNA) of constitutive NOS, i.e. neuronal NOS or endothelial NOS, was also effective in preventing the diurnal changes of TIDA neuronal activity and PRL surge at 1500 h. The same treatment of antisense ODN against the mRNA of inducible NOS, i.e. macrophage NOS, had no effect.
Progesterone (P4) has been reported to advance and augment the diurnal changes of TIDA neuronal activity and the afternoon PRL surge, by 1 h, in both proestrous and OVX+E2 rats. We further showed that l-NA dose dependently (50 but not 5 mg/kg, ip at 1000 and 1200 h) blocked the effect of P4 on TIDA neurons and serum PRL at 1300 h, which effect could be negated by simultaneous administration of l-arginine (300 mg/kg, ip). Pretreatment with antisense ODNs against the mRNA of neuronal NOS or endothelial NOS, but not macrophage NOS, was also effective in preventing the P4’s effect on TIDA neuronal activity and PRL secretion at 1300 h. In summary, NO may play a physiological role in the E2- and P4-regulated diurnal changes of TIDA neuronal activity and PRL secretion.
Chronic Exposure to High Glucose Concentrations Increases Proglucagon Messenger Ribonucleic Acid Levels and Glucagon Release from InR1G9 Cells1
Authors: Dumonteil Eric , Ritz-Laser Beate , Magnan Chistophe , Grigorescu Iléana , Ktorza Alain , Philippe Jacques 
Published: January 2014

Abstract
α cell function is impaired in diabetes. In diabetics, plasma levels of glucagon are high despite persistently elevated glucose levels and may even rise paradoxically in response to a glucose load; high plasma glucagon levels are accompanied by increased proglucagon gene expression. We have investigated the effects of high glucose concentrations on InR1G9 cells, a glucagon-producing cell line. We show here that chronically elevated glucose concentrations increase glucagon release by 2.5- to 4-fold, glucagon cell content by 2.5- to 3-fold, and proglucagon messenger RNA levels by 4- to 8-fold, whereas changes for 24 h have no effect on proglucagon messenger RNA levels. Persistently elevated glucose affects proglucagon gene expression at the level of transcription and insulin is capable of preventing this effect. We conclude that chronically elevated glucose may be an important factor in the α cell dysfunction that occurs in diabetes and thus that glucose may not only affect the β cell but also the α cell.
Pituitary and Testicular Function in Growth Hormone Receptor Gene Knockout Mice
*
Authors: Chandrashekar Varadaraj , Bartke Andrzej , Coschigano Karen T. , Kopchick John J. 
Published: January 2014

Abstract
The role of GH in the control of pituitary and testicular function is poorly understood. GH receptor gene knockout (GHR-KO) mice were recently produced. As these mice are good experimental animals to assess the influence of the effects of GH and insulin-like growth factor-I (IGF-I), the present studies were undertaken. Young adult male GHR-KO mice and their normal siblings were tested for fertility and subsequently injected (ip) with saline or GnRH (1 ng/g BW) in saline. Fifteen minutes later, blood was obtained via heart puncture. Plasma IGF-I, PRL, LH, and testosterone concentrations were measured by RIAs. In addition, the testicular testosterone response to LH treatment was evaluated in vitro. The results indicate that the absence of GH receptors (GHRs) was associated with an increase (P &lt; 0.005) in plasma PRL levels, and circulating IGF-I was not detectable. Although the basal plasma LH levels were similar in GHR-KO mice relative to those in their normal siblings, the circulating LH response to GnRH treatment was significantly (P &lt; 0.001) attenuated. Plasma testosterone levels were unaffected by disruption of the GHR gene. However, basal (P &lt; 0.01) and LH-stimulated (P &lt; 0.001) testosterone release from the isolated testes of GHR-KO mice were decreased. The rate of fertility in GHR-KO male mice was also reduced. These results indicate that the lack of GHRs (with GH resistance and lack of IGF-I secretion) induces hyperprolactinemia and alters the effect of GnRH on LH secretion as well as testicular function. Thus, GH and IGF-I influence pituitary and gonadal functions in male mice.
Pesticides Interfere with the Nongenomic Action of a Progestogen on Meiotic Maturation by Binding to its Plasma Membrane Receptor on Fish Oocytes
Authors: Das Shampa , Thomas Peter 
Published: January 2014

Abstract
Although many environmental contaminants disrupt endocrine function by binding to nuclear steroid receptors, it is not known whether they are capable of binding to steroid membrane receptors and interfering with nongenomic actions of steroids. The binding of several organochlorine pesticides to the plasma membrane receptor for the maturation-inducing steroid, 17,20β,21-trihydroxy-4-pregnen-3-one (20β-S), in the ovaries of spotted seatrout (Cynoscion nebulosus) was investigated in in vitro competition assays. Kepone and o,p′-DDD were competitive inhibitors of 20β-S binding and caused concentration-dependent displacement of [3H]-20β-S from its receptor site over the range of 10−4 to 10−6 or 10−7 M, whereas several other pesticides had lower affinities for the receptor. Interference with the nongenomic actions of 20β-S on final meiotic maturation of spotted seatrout oocytes (final oocyte maturation, FOM) was examined in an in vitro bioassay. A concentration-dependent inhibition of FOM in response to 20β-S was observed after 5 min and 12 h exposure to the same range of Kepone and o,p′-DDD concentrations (10−4 to 10−6 or 10−7 M). The close correspondence between competitive binding of the two pesticides to the 20β-S membrane receptor and their inhibition of 20β-S induced FOM suggests a mechanism of endocrine disruption mediated by binding to a steroid membrane receptor and antagonism of a nongenomic steroid action.
Glucagon-Like Peptide 1 Elevates Cytosolic Calcium in Pancreaticβ -Cells Independently of Protein Kinase A1
Authors: Bode Hans-Peter , Moormann Birgit , Dabew Regina , Göke Burkhard 
Published: January 2014

AbstractGlucagon-like peptide 1 (7–36)amide (GLP-1) is an insulinotropic intestinal peptide hormone with a potential role as antidiabetogenic therapeutic agent. It mediates a potentiation of glucose-induced insulin secretion, by activation of adenylate cyclase and subsequent elevation of cytosolic free calcium,[ Ca2+]cyt. We investigated the role of protein kinase A (PKA) in GLP-1 signal transduction, using isolated mouse islets as well as the differentiated β-cell line INS-1. Two specific inhibitors of PKA, (Rp)-adenosine cyclic 3′,5′-phosporothioate (Rp-cAMPS, up to 3 mm) and KT5720 (up to 10 μm), did not inhibit the GLP-1-induced[ Ca2+]cyt elevation. Another PKA inhibitor, H-89, reduced the [Ca2+]cyt elevation only when applied at high concentrations (10–40 μm), higher than sufficient for PKA inhibition in many cell types. Furthermore, at these concentrations, H-89 also inhibited presumably PKA-independent processes such as glucose-induced [Ca2+]cyt elevations and intracellular calcium storage. This suggests a PKA-independent action of H-89. Similarly to H-89, the potent but unselective protein kinase inhibitor staurosporine inhibited the GLP-1-induced [Ca2+]cyt elevation only at high concentrations, at which it also inhibited glucose-induced [Ca2+]cyt elevations. The same observations as with GLP-1 were made when adenylate cyclase was stimulated with forskolin, for selective examination of signal transduction downstream of receptor and G protein. Our results suggest that the GLP-1-induced[ Ca2+]cyt elevation is mediated independently of PKA and thus belongs to the yet-little-characterized ensemble of effects that are mediated by binding of cAMP to other target proteins.
Insulin-Like Growth Factor (IGF)-Binding Protein-Related Protein-1: An Autocrine/Paracrine Factor That Inhibits Skeletal Myoblast Differentiation but Permits Proliferation in Response to IGF1
Authors: Haugk Kathleen L. , Wilson Heather-Marie P. , Swisshelm Karen , Quinn LeBris S. 
Published: January 2014

AbstractSkeletal myogenic cells respond to the insulin-like growth factors (IGF-I and IGF-II) by differentiating or proliferating, which are mutually exclusive pathways. What determines which of these responses to IGF skeletal myoblast undergo is unclear. IGF-binding protein-related protein 1 (IGFBP-rP1) is a secreted protein with close homology to the IGF-binding proteins (IGFBPs) in the N-terminal region. IGFBP-rP1, previously called mac25 and IGFBP-7, is highly expressed in C2 skeletal myoblasts during the proliferative phase, but is down-regulated during myoblast differentiation. To determine the role of IGFBP-rP1 in myogenesis, IGFBP-rP1 was overexpressed in C2 myoblasts using a retroviral vector. Western blots indicated that the resulting C2-rP1 myoblasts secreted approximately 27-fold higher levels of IGFBP-rP1 than control C2-LX myoblasts that were transduced with a control vector (LXSN). Compared with C2-LX myoblasts, the differentiation responses of C2-rP1 myoblasts to IGF-I, IGF-II, insulin, and des(1–3)IGF-I were significantly reduced (P &lt; 0.05). However, proliferation responses of C2-rP1 and C2-LX myoblasts to these same factors were not significantly different. Exposure of control C2-LX myoblasts to factors secreted by C2-rP1 myoblasts using a transwell coculture system reduced C2-LX myoblast differentiation significantly (P &lt; 0.05). Experiments with the mitogen-activated protein kinase (MAPK) kinase inhibitor PD098059 suggested that IGFBP-rP1 inhibits a MAPK-dependent differentiation pathway. In confirmation of this idea, levels of phosphorylated extracellular signal-regulated kinase-2 (a MAPK) were reduced in C2-rP1 myoblasts compared with those in C2-LX myoblasts. These findings indicate that IGFBP-rP1 may function as an autocrine/paracrine factor that specifies the proliferative response to the IGFs in myogenesis.
Expression of Enzymes Synthesizing (Aldehyde Dehydrogenase 1 and Retinaldehyde Dehydrogenase 2) and Metabolizing (Cyp26) Retinoic Acid in the Mouse Female Reproductive System*
Authors: Vermot Julien , Fraulob Valérie , Dollé Pascal , Niederreither Karen 
Published: January 2014

AbstractVitamin A is required for female reproduction. Rodent uterine cells are able to synthesize retinoic acid (RA), the active vitamin A derivative, and express RA receptors. Here, we report that two RA-synthesizing enzymes [aldehyde dehydrogenase 1 (Aldh1) and retinaldehyde dehydrogenase 2 (Raldh2)] and a cytochrome P450 (Cyp26) that metabolizes vitamin A and RA into more polar metabolites exhibit dynamic expression patterns in the mouse uterus, both during the ovarian cycle and during early pregnancy. Aldh1 expression is up-regulated during diestrus and proestrus in the uterine glands, whereas Raldh2 is highly induced in the endometrial stroma in metestrus. Cyp26 expression, which is not detectable during the normal ovarian cycle, is strongly induced in the uterine luminal epithelium, 24 h after human CG hormonal administration. Raldh2 stromal expression also strongly responds to gonadotropin (PMSG and human CG) induction. Furthermore, Raldh2 expression can be hormonally induced in stromal cells of the vagina and cervix. All three enzymes exhibit differential expression profiles during early pregnancy. Aldh1 glandular expression is sharply induced at 2.5 gestational days, whereas Raldh2 stromal expression increases more steadily until the implantation phase. Cyp26 epithelial expression is strongly induced between 3.5–4.5 gestational days, i.e. when the developing blastocysts colonize the uterine lumen. These data suggest a need for precise regulation of RA synthesis and/or metabolism, in both cycling and pregnant uterus.