bluuebunny/crossref_metadata_2025_split huggingface dataset:
Tissue-Specific Transcription Start Sites and Alternative Splicing of the Parathyroid Hormone (PTH)/PTH-Related Peptide (PTHrP) Receptor Gene: A New PTH/PTHrP Receptor Splice Variant that Lacks the Signal Peptide*
Authors: Joun HoSeung , Lanske Beate , Karperien Marcel , Qian Fang , Defize Libert , Abou-Samra Abdul 
Published: January 2014

AbstractThe PTH/PTHrP receptor gene is expressed in bone and kidney as well as in many other tissues. Using primer extension followed by rapid cloning of amplified complementary DNA ends, we have isolated new PTH/PTHrP receptor complementary DNAs with different splicing patterns and have characterized a new upstream transcription start site. Three 5′ nontranslated exons, U3, U2 and U1, located 4.8, 2.5, and 1.2 kb upstream of the exon that encodes the putative signal peptide of the classical receptor (exon S), have been characterized. Four types of splicing patterns were recognized. Type I splicing pattern is transcribed from exon U1 and is spliced to exons S and E1; this pattern was found in most tissues tested. Types II, III, and IV splicing patterns are transcribed from exon U3 and have a restricted tissue distribution. Type II splice pattern, containing exons U3, U2, and S and type III splicing pattern, containing exon U3, U2, and E1 (skipping exon S), was found only in kidney. Type IV splice pattern, containing exon U3 and S was found both in kidney and ovary.Because the type III splice variant skips exon S, translation of this splice variant initiates at a different AUG codon. The type III splice variant was weakly expressed on the cell surface of COS-7 cells, as assessed by double antibody binding assay, and no detectable ligand binding was observed on intact cells. The type III splice variant, however, increased cAMP accumulation in COS-7 cells when challenged with PTH(1–34), PTH(1–84) and hPTHrP(1–36) with EC50s that are similar to those observed in COS-7 cells expressing the type I variant but with a maximum stimulation that was lower than that observed in COS-7 cells expressing the type I variant. These data indicate low levels of cell surface expression of the type III splice variant. Treatment of COS-7 cells with tunicamycin decreased the size of the type I splice variant from a broad band of 85 kDa to a compact band of about 60 kDa. The type III splice variant did not change in size in COS-7 cells treated with tunicamycin, indicating that the type III splice variant did not undergo any glycosylation step. In conclusion, the PTH/PTHrP receptor gene uses alternate promoters in a tissue-specific manner that results in several tissue-specific alternatively spliced transcripts. One of these transcripts, the type III splice variant, is expressed in kidney and lacks the signal peptide.
Galparan: A Powerful Insulin-Releasing Chimeric Peptide Acting at a Novel Site*
Authors: Östenson Claes-Göran , Zaitsev Sergei , Berggren Per-Olof , Efendic Suad , Langel Ülo , Bartfai Tamas 
Published: January 2014

AbstractGalparan is a 27-amino acid long chimeric peptide, GWTLNSAGYLLGP-INLKALAALAKKIL amide, consisting of galanin-(1–13) linked to mastoparan amide via a peptide bond to provide the mastoparan and galanin effector parts of the molecules. Galparan (10μ m) powerfully stimulates insulin secretion from isolated rat pancreatic islets in a reversible and dose-dependent manner; the stimulation is 26-fold at 3.3 mm glucose and 6-fold at 16.7 mm glucose. Galparan also enhances insulin secretion to a similar extent from islets of diabetic GK rats. The stimulatory effect of galparan on insulin release is not directly dependent on extracellular Ca2+, nor can it be explained only by changes in free cytosolic Ca2+ concentrations. Furthermore, galparan is effective in evoking insulin release in B cells depolarized by 25 mm KCl when ATP-sensitive K+ channels are kept open by diazoxide. Thus, galparan, like mastoparan, stimulates exocytosis of insulin at a distal site in the stimulus-secretion coupling of the B cell. This distal site is not identical to that used by mastoparan, as pertussis toxin pretreatment does not influence the insulinogenic effect of galparan. In conclusion, galparan evokes a large and reversible insulin secretion, acting at a yet unknown distal site and also promoting exocytosis in depolarized B cells from normal rats as well as diabetic GK rats.
Interaction of Mouse Placental Lactogens and Androgens in Regulating Progesterone Release in Cultured Mouse Luteal Cells
Authors: Thordarson G. , Galosy S. , Gudmundsson G. O. , Newcomer B. , Sridaran R. , Talamantes F. 
Published: January 2014

Abstract
Pituitary hormones are essential for the maintenance of the corpus luteum in the pregnant mouse during the first half of gestation. Thereafter, hormones from the placenta take over the luteotropic role of the pituitary hormones. Mouse placental lactogen-I (mPL-I) and mPL-II, two PRL-like hormones produced in the placenta, are probably necessary for the maintenance of the corpus luteum in the latter half of pregnancy. A culture system of luteal cells from pregnant mice was developed to investigate the role of hormones from the placenta that may be important for the function of the corpus luteum. Mice were killed on days 10, 14, and 18 of pregnancy, and the corpora lutea were excised from the ovaries and digested in 0.1% collagenase, 0.002% DNase for 1 h. The resulting luteal cell suspension was plated onto 96-well plates coated with fibronectin (1 × 105 cells/well) and cultured for 1–3 days. Medium was changed daily. The cells were treated with various concentrations and combinations of mPL-I, mPL-II, mouse PRL, androstenedione, dihydrotestosterone, 17β-estradiol (E2), testosterone, hydroxyflutamide, cycloheximide, actinomycin D, and fadrozole to study the effects of these different treatments on progesterone (P4) production. The three lactogens (mPL-I, mPL-II, and mouse PRL) all stimulated the release of P4 from the luteal cells. The potency of the lactogens was similar and did not depend on the stage of pregnancy at which the luteal tissue was obtained. However, the responsiveness of the cells to all hormone-stimulated P4 release was gradually reduced the later in pregnancy the tissue was collected. Androgens also stimulated the release of P4 from the luteal cells, and when administered together, the lactogens and the androgens acted synergistically to stimulate P4 release. The androgens acted directly but not through conversion to E2, as determined by the findings that 1) the effects of the androgens could not be reproduced by E2 administration, 2) nonaromatizable androgen dihydrotestosterone was as effective as aromatizable androgens, and 3) aromatase inhibitor did not prevent the action of the androgens to stimulate the P4 release. The effect of the androgens on the P4 release was rapid, occurring within 15 min of hormone administration. It was not prevented by inhibitors of protein and RNA synthesis, and the intracellular androgen receptor antagonist hydroxyflutamide did not affect the androgen action. Therefore, the androgen effects were not mediated through the intracellular androgen receptor and de novo protein synthesis was not needed for androgen-stimulated P4 release.
A Steroidogenic Factor-1-Binding Site and Cyclic Adenosine 3′,5′-Monophosphate Response Element-Like Elements Are Required for the Activity of the Rat Aromatase Promoter in Rat Leydig Tumor Cell Lines**This work was completed while M.Y. was a member of the Cell Regulation Graduate Program at the University of Texas Southwestern Medical Center. This work was supported by NIH Grant DK-03892.
Authors: Young Maureen , McPhaul Michael J. 
Published: January 2014

AbstractAlthough transcription initiation within CYP19 (cytochrome P450 aromatase) occurs immediately 5′ to the initiator methionine (proximal promoter) in two rat Leydig tumor cell lines (R2C and H540) that express high aromatase activity and in rat ovary, the patterns of aromatase expression in the two cell types are distinctive. To define mechanisms controlling different patterns of expression of the rat aromatase proximal promoter, we performed transient transfection and gel mobility shift assays. Transfection experiments using different sized promoter fragments fused to a reporter gene were used to identify regions that are functionally important for transcriptional regulation in steroidogenic cell lines [R2C, H540, and Y1 (mouse adrenocortical cells that express low aromatase activity)]. These experiments indicate that the cAMP response element (CRE) at −231 and the steroidogenic factor-1 (SF1) motif are both required for expression of the reporter gene in each steroidogenic cell line and that the CRE at− 169 is similarly required in R2C cells. Gel mobility shift assays confirm binding of nuclear proteins from the steroidogenic cell lines to the SF1 motif and to CRE (−231). Leydig tumor cells also contain nuclear proteins that bind to the CRE (−169), but nuclear extracts from R2C cells produce a uniquely shifted band compared with H540 cells. These results suggest that differences in proteins that bind to distinct elements within the rat aromatase promoter may be responsible for different patterns and levels of aromatase expression in these steroidogenic cell lines.
A Phospholipase A2-Related Snake Venom (from Crotalus durissus terrificus) Stimulates Neuroendocrine and Immune Functions: Determination of Different Sites of Action*
Authors: Chisari Andrea , Spinedi Eduardo , Voirol Marie-Jeanne , Giovambattista Andrés , Gaillard Rolf C. 
Published: January 2014

Abstract
Immune neuroendocrine interactions are vital for the individual’s survival in certain physiopathological conditions, such as sepsis and tissular injury. It is known that several animal venoms, such as those from different snakes, are potent neurotoxic compounds and that their main component is a specific phospholipase A type 2 (PLA2). It has been described recently that the venom from Crotalus durissus terrificus [snake venom (SV), in the present study] possesses some cytotoxic effect in different in vitro and in vivo animal models. In the present study, we investigated whether SV and its main component, PLA2 (obtained from the same source), are able to stimulate both immune and neuroendocrine functions in mice, thus characterizing this type of neurotoxic shock. For this purpose, several in vivo and in vitro designs were used to further determine the sites of action of SV-PLA2 on the hypothalamo-pituitary-adrenal (HPA) axis function and on the release of the pathognomonic cytokine, tumor necrosis factor α (TNFα), of different types of inflammatory stress. Our results indicate that SV (25 μg/animal) and PLA2 (5 μg/animal), from the same origin, stimulate the HPA and immune axes when administered (ip) to adult mice; both preparations were able to enhance plasma glucose, ACTH, corticosterone (B), and TNFα plasma levels in a time-related fashion. SV was found to activate CRH- and arginine vasopressin-ergic functions in vivo and, in vitro, SV and PLA2 induced a concentration-related (0.05–10 μg/ml) effect on the release of both neuropeptides. SV also was effective in changing anterior pituitary ACTH and adrenal B contents, also in a time-dependent fashion. Direct effects of SV and PLA2 on anterior pituitary ACTH secretion also were found to function in a concentration-related fashion (0.001–1 μg/ml), and the direct corticotropin-releasing activity of PLA2 was additive to those of CRH and arginine vasopressin; the corticotropin-releasing activity of both SV and PLA2 were partially reversed by the specific PLA2 inhibitor, manoalide. On the other hand, neither preparation was able to directly modify spontaneous and ACTH-stimulated adrenal B output. The stimulatory effect of SV and PLA2 on in vivo TNFα release was confirmed by in vitro experiments on peripheral mononuclear cells; in fact, both PLA2 (0.001–1 μg/ml) and SV (0.1–10 μg/ml), as well as concavalin A (1–100 μg/ml), were able to stimulate TNFα output in the incubation medium.
Our results clearly indicate that PLA2-dependent mechanisms are responsible for several symptoms of inflammatory stress induced during neurotoxemia. In fact, we found that this particular PLA2-related SV is able to stimulate both HPA axis and immune functions during the acute phase response of the inflammatory processes.
Neutralization of Relaxin within the Brain Affects the Timing of Birth in Rats*
Authors: Summerlee A. J. S. , Ramsey D. G. , Poterski R. S. 
Published: January 2014

AbstractExperiments were performed to determine whether neutralization of relaxin in the brain, by injecting monoclonal antibodies to rat relaxin into the ventricular system of the brain, affected either the timing or the processes of birth in rats.Pregnant rats were injected daily through a chronically implanted intracerebroventricular cannula either with a specific monoclonal antibody raised against rat relaxin from days 12–22 of gestation or with an antibody raised against fluorescein as a control. The rats were watched closely from the afternoon of day 20 of pregnancy, and the process of birth was observed. No sign of dystocia was observed in any of the rats in the experiment. Neutralization of endogenous relaxin caused a significant decrease in the length of gestation (505.4 ± 3.1 h) compared with that in rats treated with PBS (524.6 ± 0.5 h) or that in rats treated with a nonspecific antibody (525.9 ± 0.7 h). The time to the onset of delivery was also shorter in the relaxin-neutralized group (507.8 ± 1.1 h) compared with that in either PBS-treated (526.5 ± 0.6 h) or fluorescein antibody-treated (525.3 ± 0.7 h) animals. In contrast, there was no significant effect of the relaxin antibody on length of straining, duration of parturition, delivery interval, live birth rate, or body weight of the neonates. Premature delivery in the relaxin-neutralized group was accompanied by a 24-h advance in the fall in plasma progesterone.These data support the hypothesis that there may be a central relaxin system that is independent of the peripheral relaxin system. Central relaxin may have a significant physiological role on the timing of pregnancy in the rat, but does not affect the course of labor once it has started.
The Synergistic Effects of Vitamin D Metabolites and Transforming Growth Factor-β on Costochondral Chondrocytes Are Mediated by Increases in Protein Kinase C Activity Involving Two Separate Pathways*
Authors: Schwartz Z. , Sylvia V. L. , Dean D. D. , Boyan B. D. 
Published: January 2014

AbstractTransforming growth factor-β (TGFβ), as well as the vitamin D3 metabolites 1,25-dihydroxyvitamin D3 (1,25) and 24,25-dihydroxyvitamin D3 (24,25), regulate chondrocyte differentiation and maturation during endochondral bone formation. Both the growth factor and secosteroids also affect protein kinase C (PKC) activity, although each has its own unique time course of enzyme activation. Vitamin D3 metabolite effects are detected soon after addition to the media, whereas TGFβ effects occur over a longer term. The present study examines the interrelation between the effects of 1,25, 24,25, and TGFβ on chondrocyte differentiation, matrix production, and proliferation. We also examined whether the effect is hormone-specific and maturation-dependent and whether the effect of combining hormone and growth factor is mediated by PKC.This study used a chondrocyte culture model developed in our laboratory that allows comparison of chondrocytes at two stages of differentiation: the more mature growth zone (GC) cells and the less mature resting zone chondrocyte (RC) cells. Only the addition of 24,25 with TGFβ showed synergistic effects on RC alkaline phosphatase-specific activity (ALPase). No similar effect was found when 24,25 plus TGFβ was added to GC cells or when 1,25 plus TGFβ were added to GC or RC cells. The addition of 1,25 plus TGFβ and 24,25 plus TGFβ to GC and RC cells, respectively, produced a synergistic increase in [35S]sulfate incorporation and had an additive effect on [3H]thymidine incorporation. To examine the signal transduction pathway involved in producing the synergistic effect of 24,25 and TGFβ on RC cells, the level of PKC activity was examined. Addition of 24,25 and TGFβ for 12 h produced a synergistic increase in PKC activity. Moreover, a similar effect was found when 24,25 was added for only the last 90 min of a 12-h incubation. However, a synergistic effect could not be found when 24,25 was added for the last 9 min or the first 90 min of incubation. To further understand how 24,25 and TGFβ may mediate the observed synergistic increase in PKC activity, the pathways potentially leading to activation of PKC were examined. It was found that 24,25 affects PKC activity through production of diacylglycerol, not through activation of G protein, whereas TGFβ only affected PKC activity through G protein.The results of the present study indicate that vitamin D metabolites and TGFβ produced a synergistic effect that is maturation-dependent and hormone-specific. Moreover, the synergistic effect between 24,25 and TGFβ was mediated by activation of PKC through two parallel pathways: 24,25 through diacylglycerol production and TGFβ through G protein activation.
The First Extracellular Domain of Corticotropin Releasing Factor-R1 Contains Major Binding Determinants for Urocortin and Astressin*
Authors: Perrin Marilyn H. , Sutton Steve , Bain Deborah L. , Berggren W. Travis , Vale Wylie W. 
Published: January 2014

AbstractThe CRF receptors are members of a 7-transmembrane receptor family that includes GH-releasing hormone (GRF), calcitonin, vasoactive intestinal peptide (VIP), secretin, and PTH receptors. To determine the structural features of the CRF receptor that may influence ligand recognition, a series of mutant receptors was analyzed for binding to astressin, a CRF antagonist, and to urocortin, a CRF agonist. Mutant receptors included chimeras between the CRF-R1 and GRF-R or Activin IIB-R, a single membrane spanning receptor serine/threonine kinase. Binding to the mutant receptors was assessed using 125I-[DTyr1] astressin (Ast*) and 125I-[Tyr0]-rat urocortin (Ucn*). There was no binding to a chimeric receptor in which the first extracellular domain (E1c) (i.e. the N-terminal region) of the CRF-R1 was replaced by that of the GRF-R. The complementary chimera in which E1 domain of the GRF-R was replaced by that of the CRF-R1 bound astressin and urocortin with Ki values approximately 10 nm, compared with inhibitory binding dissociation constant (Ki) values of approximately 2–4 nm for the wild-type CRF-R1. The chimera in which E1 of the activin IIB receptor was replaced by E1 of the CRF-R1 bound astressin with a Ki approximately 4 nm. A chimera in which both the first and fourth extracellular domains of the CRF-R1 replaced the corresponding domains of the GRF-R bound astressin with Ki approximately 4 nm and urocortin with a Ki approximately 2 nm. A chimera in which all four extracellular domains of the CRF receptor replaced those of the GRF-R bound astressin and urocortin with Ki values approximately 4 nm and approximately 1 nm, respectively. In conclusion, the major determinants for high affinity binding of CRF agonists and antagonists to CRF-R1 are found in the first extracellular domain of the receptor.
Overexpression of Insulin-Like Growth Factor-Binding Protein-4 (IGFBP-4) in Smooth Muscle Cells of Transgenic Mice through a Smooth Muscle α-Actin-IGFBP-4 Fusion Gene Induces Smooth Muscle Hypoplasia
Authors: Wang Jianwei , Niu Wen , Witte David P. , Chernausek Steven D. , Nikiforov Yuri E. , Clemens Thomas L. , Sharifi Behrooz , Strauch Arthur R. , Fagin James A. 
Published: January 2014

AbstractInsulin-like growth factor I (IGF-I) has been postulated to function as a smooth muscle cell (SMC) mitogen and to play a role in the pathogenesis of bladder hypertrophy, estrogen-induced uterine growth, and restenosis after arterial angioplasty. IGF-binding protein-4 (IGFBP-4) inhibits IGF-I action in vitro and is the most abundant IGFBP in the rodent arterial wall. To explore the function of this binding protein in vivo, transgenic mouse lines were developed harboring fusion genes consisting of a rat IGFBP-4 complementary DNA cloned downstream of either a −724 bp fragment of the mouse smooth muscle α-actin 5′-flanking region (SMP2-BP-4) or− 1074 bp, 63 bp of 5′-untranslated region, and 2.5 kb of intron 1 of smooth muscle α-actin (SMP8-BP-4). SMP2-BP-4 mice expressed low levels of the exogenous IGFBP-4 messenger RNA (mRNA), which was not specifically targeted to SMC-rich tissue environments, and were therefore not analyzed further. Six SMP8-BP-4 transgenic lines derived from separate founders were characterized. Mating of hemizygous SMP8-BP-4 mice with controls produced about 50% transgenic offspring, with equal sex distribution. Expression of IGFBP-4 mRNA in nontransgenic littermates was maximal in liver and kidney. By contrast, transgenic IGFBP-4 mRNA expression, distinguished because of a smaller transcript size, was confined to SMC-containing tissues, with the following hierarchy: bladder &gt; aorta &gt; stomach = uterus. There was no transgene expression in skeletal muscle, brain, or cardiac myocytes. The abundance of IGFBP-4 measured by Western ligand blotting or by immunoblotting, was 8- to 10-fold higher in aorta and bladder of SMP8-BP-4 mice than in their nontransgenic littermates, with no change in plasma IGFBP-4 levels. Transgenic mice exhibited a significant reduction in wet weight of SMC-rich tissues, including bladder, intestine, aorta, uterus, and stomach, with no change in total body or carcass weight. In situ hybridization showed that transgene expression was targeted exclusively to the muscular layers of the arteries, veins, bladder, ureter, stomach, intestine, and uterus. Overexpression of IGFBP-4 was associated with SMC hypoplasia, a reciprocal phenotype to that of transgenic mice overexpressing IGF-I under control of the same promoter (SMP8-IGF-I). Double transgenic mice derived from mating SMP8-BP-4 with SMP8-IGF-I animals showed a modest decrease in wet weight at selected SMC tissues. Although we cannot exclude that the effects of IGFBP-4 may be IGF independent, these data suggest that IGFBP-4 is a functional antagonist of IGF-I action on SMC in vivo.