bluuebunny/crossref_metadata_2025_split huggingface dataset:
Characterization of a Propylthiouracil-Insensitive Type I Iodothyronine Deiodinase*
Authors: Sanders Jo P. , Van der Geyten Serge , Kaptein Ellen , Darras Veerle M. , Kühn Eduard R. , Leonard Jack L. , Visser Theo J. 
Published: January 2014

AbstractMammalian type I iodothyronine deiodinase (D1) activates and inactivates thyroid hormone by outer ring deiodination (ORD) and inner ring deiodination (IRD), respectively, and is potently inhibited by propylthiouracil (PTU). Here we describe the cloning and characterization of a complementary DNA encoding a PTU-insensitive D1 from teleost fish (Oreochromis niloticus, tilapia). This complementary DNA codes for a protein of 248 amino acids, including a putative selenocysteine (Sec) residue, encoded by a TGA triplet, at position 126. The 3′ untranslated region contains two putative Sec insertion sequence (SECIS) elements. Recombinant enzyme expressed in COS-1 cells catalyzes both ORD of T4 and rT3 and IRD of T3 and T3 sulfate with the same substrate specificity as native tilapia D1 (tD1), i.e. rT3 ≫ T4 &gt; T3 sulfate &gt; T3. Native and recombinant tD1 show equally low sensitivities to inhibition by PTU, iodoacetate, and gold thioglucose compared with the potent inhibitions observed with mammalian D1s. Because the residue 2 positions downstream from Sec is Pro in tD1 and in all (PTU-insensitive) type II and type III iodothyronine deiodinases but Ser in all PTU-sensitive D1s, we prepared the Pro128Ser mutant of tD1. The mutant enzyme showed strongly decreased ORD and somewhat increased IRD activity, but was still insensitive to PTU. These results provide new information about the structure-activity relationship of D1 concerning two characteristic properties, i.e. catalysis of both ORD and IRD, and inhibition by PTU.
Transforming Growth Factor-β1 Induces a Mesenchyme-Like Cell Shape without Epithelial Polarization in Thyrocytes and Inhibits Thyroid Folliculogenesis in Collagen Gel Culture
Authors: Toda Shuji , Matsumura Sueo , Fujitani Noboru , Nishimura Tomohisa , Yonemitsu Nobuhisa , Sugihara Hajime 
Published: January 2014

AbstractTransforming growth factor-β1 (TGFβ1) induces a mesenchyme-like cell shape in some epithelial cell types. To clarify the role of TGFβ1 in the morphological regulation of thyrocytes, we performed collagen gel culture of porcine thyrocytes with serum-free medium. TGFβ1-nontreated cells organized follicles. In contrast, the cells treated with 10 ng/ml TGFβ1 became spindle shaped, i.e. they resembled mesenchymal fibroblasts, and did not form follicles. To characterize the spindle-shaped cells, we examined the fine structures and expression of thyroglobulin (Tg) and cytoskeletal proteins using electron microscopy, immunohistochemistry, and immunoblotting. TGFβ1-nontreated cells had microvilli at the apical side facing follicle lumen and had basal lamina at the basal side in contact with collagen gel. TGFβ1-treated cells showed both microvilli and basal lamina at the basal side. TGFβ1-nontreated cells expressed Tg, whereas TGFβ1-treated cells showed no expression. TGFβ1-nontreated cells barely expressed vimentin, but they expressed enough cytokeratin. TGFβ1-treated cells extensively displayed vimentin along with the change in shape to become spindle-like and retained a decreased expression of cytokeratin. TSH (10 mU/ml) did not essentially influence any TGFβ1 effects on the cells. These results indicate that TGFβ1 induces a mesenchyme-like cell shape accompanied by cytoskeletal molecular change and the loss of both epithelial polarization and a function in thyrocytes, and that it results in inhibiting thyroid folliculogenesis with or without TSH.
The Role of the Active Site Cysteine in Catalysis by Type 1 Iodothyronine Deiodinase*
Authors: Sun Ben C. , Harney John W. , Berry Marla J. , Larsen P. Reed 
Published: January 2014

AbstractType 1 iodothyronine deiodinase (deiodinase 1) is a selenoenzyme that converts the prohormone T4 to the active thyroid hormone T3 by outer ring deiodination or to the inactive metabolite rT3 by inner ring deiodination. Although selenocysteine has been demonstrated to be essential for the biochemical profile of deiodinase 1, the role of a highly conserved, active site cysteine (C124 in rat deiodinase 1) has not been defined. The present studies examined the effects of a Cys124Ala mutation on rat deiodinase 1 enzymatic function and substrate affinity. At a constant 10-mm concentration of dithiothreitol (DTT), the C124A mutant demonstrated a 2-fold lower apparent maximal velocity (Vmax) and Km for rT3 (KmrT3) than the wild type for outer ring deiodination, whereas the Vmax/Km ratio was unchanged. Similarly, the apparent Vmax and KmT3 sulfate for inner ring deiodination were 2-fold lower in the C124A mutant relative to those in the wild type, with no change in the Vmax/Km ratio. The C124A mutant exhibited ping-pong kinetics in the presence of DTT, and substitution of the active site cysteine increased the KmDTT by 14-fold relative to that of the wild-type enzyme, with no significant effects on KmrT3 or Vmax. The C124A mutant was inhibited by propylthiouracil in an uncompetitive fashion and exhibited a 2-fold increase in Kipropylthiouracil compared with that of the wild type. KmrT3 was also reduced for the C124A mutant when 5 mm reduced glutathione, a potential physiological monothiol cosubstrate, was used in outer ring deiodination assays. These results demonstrate that thiol cosubstrate interactions with C124 in type 1 deiodinase play an important role in enhancing catalytic efficiency for both outer and inner ring deiodination.
Cloning, Novel Promoter Sequence, and Estrogen Regulation of a Rat Oxytocin Receptor Gene*
Authors: Bale Tracy L. , Dorsa Daniel M. 
Published: January 2014

AbstractExpression of the oxytocin receptor (OR) gene in vivo is known to be regulated by estradiol (E2). We have cloned and sequenced 4 kilobases (kb) of 5′-flanking DNA of the rat OR gene and identified an internal segment of 1260 nucleotides that was absent in an initial publication of this promoter and an additional 2 kb of upstream sequence. This novel internal region is located between two large tg nucleotide repeats. PCR amplification using genomic DNA verified that this sequence is present in the rat genome. To explain transcriptional effects of E2, a palindromic estrogen response element (ERE) that is active in estrogen receptor binding was identified within this new sequence, approximately 4 kb 5′ of the translational start site. The ability of E2 to enhance transcription of this promoter was tested in transfection experiments in MCF7 cells. E2 only weakly induced transcription of a truncated construct. Mutational analysis of the ERE in the context of a basal promoter indicated that it functions as an enhancer, and that mutation of two bases eliminates this activity. Further support of the efficacy of this response was shown in mobility gel shift assays in which the OR ERE bound estrogen receptor present in uterine extracts. Receptor binding studies using 125I-ornithine vasotocin in MCF7 cells revealed that E2 dramatically up-regulated endogenous ORs. Western blot analysis confirmed this increase in OR protein with E2 treatment of MCF7 cells. These studies have identified a novel region of the rat OR promoter containing an upstream palindromic ERE that imparts E2 inducibility of OR gene transcription.
The Effects of Orbital Spaceflight on Bone Histomorphometry and Messenger Ribonucleic Acid Levels for Bone Matrix Proteins and Skeletal Signaling Peptides in Ovariectomized Growing Rats*
Authors: Cavolina Jason M. , Evans Glenda L. , Harris Steven A. , Zhang Minzhi , Westerlind Kim C. , Turner Russell T. 
Published: January 2014

Abstract
A 14-day orbital spaceflight was performed using ovariectomized Fisher 344 rats to determine the combined effects of estrogen deficiency and near weightlessness on tibia radial bone growth and cancellous bone turnover. Twelve ovariectomized rats with established cancellous osteopenia were flown aboard the space shuttle Columbia (STS-62). Thirty ovariectomized rats were housed on earth as ground controls: 12 in animal enclosure modules, 12 in vivarium cages, and 6 killed the day of launch for baseline measurements. An additional 18 ovary-intact rats were housed in vivarium cages as ground controls: 8 rats were killed as baseline controls and the remaining 10 rats were killed 14 days later. Ovariectomy increased periosteal bone formation at the tibia-fibula synostosis; cancellous bone resorption and formation in the secondary spongiosa of the proximal tibial metaphysis; and messenger RNA (mRNA) levels for the prepro-α2(1) subunit of type 1 collagen, osteocalcin, transforming growth factor-β, and insulin-like growth factor I in the contralateral proximal tibial metaphysis and for the collagen subunit in periosteum pooled from tibiae and femora and decreased cancellous bone area. Compared to ovariectomized weight-bearing rats, the flight group experienced decreases in periosteal bone formation, collagen subunit mRNA levels, and cancellous bone area. The flight rats had a small decrease in the cancellous mineral apposition rate, but no change in the calculated bone formation rate. Also, spaceflight had no effect on cancellous osteoblast and osteoclast perimeters or on mRNA levels for bone matrix proteins and signaling peptides. On the other hand, spaceflight resulted in an increase in bone resorption, as ascertained from the diminished retention of a preflight fluorochrome label. This latter finding suggests that osteoclast activity was increased. In a follow-up ground-based experiment, unilateral sciatic neurotomy of ovariectomized rats resulted in cancellous bone loss in the unloaded limb in excess of that induced by gonadal hormone deficiency. This additional bone loss was arrested by estrogen replacement. We conclude from these studies that estrogen alters the expression of signaling peptides believed to mediate skeletal adaptation to changes in mechanical usage and likewise modifies the skeletal response to mechanical unloading.
Characterization of the Cholecystokinin and Gastrin Genes from the Bullfrog, Rana catesbeiana: Evolutionary Conservation of Primary and Secondary Sites of Gene Expression
Authors: Rourke Ian J. , Rehfeld Jens F. , Møller Morten , Johnsen Anders H. 
Published: January 2014

AbstractThe gastrin and cholecystokinin (CCK) genes, and the complementary DNAs they encode, have been isolated and sequenced from the bullfrog, Rana catesbeiana. The CCK gene promoter region possess the same four well characterized transcriptional control elements as the human CCK gene, namely an E-box, AP-1 binding site, Sp1 site, and TATA box. In contrast, no obvious regulatory motifs are conserved in the gastrin gene. Alignment of the bullfrog preprohormone sequences with other members of the CCK/gastrin peptide family showed that preproCCK has been conserved to a greater degree during evolution than preprogastrin. In mammalian species, gastrin gene expression is typically associated with the antrum, and CCK with the small intestine and brain. However numerous secondary sites of CCK/gastrin gene expression have also been found. RT-PCR showed a high degree of conservation of both primary and secondary sites of CCK/gastrin production between mammals and the bullfrog, with gastrin messenger RNA being detected in the antrum, duodenum, colon, pancreas, brain, and testes, whereas CCK mRNA was observed in the brain, lung, testes, and throughout the length of the small intestine. In situ hybridization using radiolabeled gene specific antisense oligonucleotides uncovered CCK and gastrin messenger RNA in distinct areas of the bullfrog central nervous system and pituitary gland. Notably, the gastrin gene was expressed in the pituitary gland and hypothalamus of the bullfrog, as previously seen in mammals. This highly preserved tissue expression pattern suggests that gastrin plays specific roles in the hypothalamus and pituitary gland that are distinct from those of CCK. Our findings show that in spite of the structural resemblance, bullfrog CCK and gastrin constitute independent neuroendocrine peptide systems.
Retinoid X Receptors in the Kidney: Their Protein Expression and Functional Significance
Authors: Sugawara Akira , Sanno Naoko , Takahashi Nobuyuki , Osamura R. Yoshiyuki , Abe Keishi 
Published: January 2014

AbstractRetinoid X receptors (RXRs) heterodimerize with 1,25-dihydroxyvitamin D3 (VD) receptor (VDR), and play important roles in VD-regulated transactivation. VD acts on many tissues including kidney for the regulation of calcium homeostasis. In the kidney, the expression of VDR in the tubular cells has been well studied. In contrast, little is known about the localization and the functional significance of RXRs there. In order to elucidate these questions, we first performed immunohistochemical analyses of rat kidney using isoform-specific antimouse RXR antibodies we have previously reported. Interestingly, all RXR isoforms, predominantly RXRα, mainly localized to the proximal and the distal tubules, but not to the glomeruli. The serial section staining using anti-VDR antibody showed the colocalization of RXRα and VDR in those tubular cells. In order to elucidate the functional significance of endogenous receptors in the tubular cells, we next performed transient transfection studies using the tubular-cell derived Madin-Darby bovine kidney cells, which express both endogenous VDR and RXR. We transfected a reporter plasmid containing direct repeat 3 (DR3) sequence, to which only RXR/VDR heterodimer can bind, and found that VD and 9-cis retinoic acid, as well as VD and RXR selective agonist LG100153, had an additive effect for the DR3 transactivation. Taken together, we speculate that endogenous RXRs co-localize with VDR, and coregulate VD-dependent genes in the tubular cells of the kidney as RXR/VDR heterodimer.
A Protective Role for Heme Oxygenase Expression in Pancreatic Islets Exposed to Interleukin-1β**This work was supported by NIH Grant DK-25705 (to S.G.L.).
Authors: Ye Jing , Laychock Suzanne G. 
Published: January 2014

AbstractHeme oxygenase (HO)-1 expression was investigated in rat isolated pancreatic islets. Freshly isolated islets showed no evidence of HO-1 expression. After a 20-h culture, there was a small increase in HO-1 in control islets, and interleukin-1β (IL-1β) induced HO-1 expression above control levels. NG-monomethyl-l-arginine inhibited the IL-1β-induced increase in HO-1. Sodium nitroprusside-generated nitric oxide also increased HO-1 expression. CoCl2 induced a concentration- and time-dependent increase in HO-1, but not heat shock protein 70, expression. Cobalt chloride (CoCl2) protected islets from the inhibitory effects of IL-1β on glucose-stimulated insulin release and glucose oxidation. Nickel chloride did not mimic the effects of CoCl2. An inhibitor of HO-1 activity, zinc-protoporphyrin IX (ZnPP), prevented the protective effect of CoCl2 on insulin release with IL-1β but did not affect HO-1 expression or the inhibitory response to IL-1β alone. ZnPP also inhibited the protective effect of hemin in IL-1β-treated islets. CoCl2 inhibited the marked increase in islet nitrite production in response to IL-1β. Cobalt-protoporphyrin IX (CoPP), which increased HO expression and activity, also protected islets from the inhibitory effects of IL-1β, even though IL-1β largely blocked the CoPP-induced increase in HO-1 expression. In βHC9 cells, CoCl2 increased HO-1 expression and HO activity, whereas CoPP directly activated HO. ZnPP inhibited basal and CoCl2-stimulated HO activity. Thus, increased HO-1 expression and/or HO activity in response to CoCl2, CoPP, and hemin, seems to mediate protective responses of pancreatic islets against IL-1β. HO-1 may be protective of β-cells because of the scavenging of free heme, the antioxidant effects of the end-product bilirubin, or the generation of carbon monoxide, which might have insulin secretion-promoting effects and inhibitory effects on nitric oxide synthase.
Exendin-(9–39) Is an Inverse Agonist of the Murine Glucagon-Like Peptide-1 Receptor: Implications for Basal Intracellular Cyclic Adenosine 3′,5′-Monophosphate Levels and β-Cell Glucose Competence**This work was supported by Grant 31-46958.96 from the Swiss National Science Foundation (to B.T.).
Authors: Serre Véronique , Dolci Wanda , Schaerer Elizabeth , Scrocchi Louise , Drucker Daniel , Efrat Shimon , Thorens Bernard 
Published: January 2014

AbstractThe effect of exendin-(9–39), a described antagonist of the glucagon-like peptide-1 (GLP-1) receptor, was evaluated on the formation of cAMP- and glucose-stimulated insulin secretion (GSIS) by the conditionally immortalized murine βTC-Tet cells. These cells have a basal intracellular cAMP level that can be increased by GLP-1 with an EC50 of approximately 1 nm and can be decreased dose dependently by exendin-(9–39). This latter effect was receptor dependent, as aβ -cell line not expressing the GLP-1 receptor was not affected by exendin-(9–39). It was also not due to the endogenous production of GLP-1, because this effect was observed in the absence of detectable preproglucagon messenger RNA levels and radioimmunoassayable GLP-1. Importantly, GSIS was shown to be sensitive to this basal level of cAMP, as perifusion of βTC-Tet cells in the presence of exendin-(9–39) strongly reduced insulin secretion. This reduction of GSIS, however, was observed only with growth-arrested, not proliferating, βTC-Tet cells; it was also seen with nontransformed mouse β-cells perifused in similar conditions. These data therefore demonstrated that 1) exendin-(9–39) is an inverse agonist of the murine GLP-1 receptor; 2) the decreased basal cAMP levels induced by this peptide inhibit the secretory response of βTC-Tet cells and mouse pancreatic islets to glucose; 3) as this effect was observed only with growth-arrested cells, this indicates that the mechanism by which cAMP leads to potentiation of insulin secretion is different in proliferating and growth-arrested cells; and 4) the presence of the GLP-1 receptor, even in the absence of bound peptide, is important for maintaining elevated intracellular cAMP levels and, therefore, the glucose competence of theβ -cells.
Role of Nuclear Factor-κB Activation in Cytokine- and Sphingomyelinase-Stimulated Inducible Nitric Oxide Synthase Gene Expression in Vascular Smooth Muscle Cells**This work was supported in part by grants-in-aid from the Ministry of Education, Science, and Culture and the Ministry of Health and Welfare of Japan.
Authors: Katsuyama Koichi , Shichiri Masayoshi , Marumo Fumiaki , Hirata Yukio 
Published: January 2014

AbstractInflammatory cytokines, such as interleukin-1β (IL-1β) and tumor necrosis factor-α (TNFα), are known to activate sphingomyelinase (SMase) and nuclear factor-κB (NF-κB) in certain cell types, which also stimulate inducible nitric oxide synthase (iNOS) gene in vascular smooth muscle cells (VSMCs). However, it remains unknown whether the SMase pathway is involved in iNOS gene expression in VSMCs. Therefore, the present study was designed to examine whether SMase induces iNOS gene expression via the NF-κB activation pathway similar to that of IL-1β and TNFα in cultured rat VSMCs. Neutral SMase, although less potently than IL-1β and TNFα, stimulated nitrite/nitrate (NOx) production, and iNOS messenger RNA and protein expression, as assessed by Northern and Western blot analyses, respectively. Neutral SMase, IL-1β, and TNFα activated NF-κB, as revealed by electrophoretic mobility shift assay, and its nuclear translocation, as demonstrated by immunocytochemical study. Neutral SMase potentiated NOx production, iNOS expression, and NF-κB activation stimulated by TNFα, but not by IL-1β. Aldehyde peptide proteasome inhibitors completely blocked NOx production, iNOS expression, NF-κB activation, and its nuclear translocation induced by cytokines and neutral SMase. IL-1β and TNFα, but not neutral SMase, caused a transient decrease in IκB-α protein levels, whereas IκB-β protein expression was not affected by either agent. Proteasome inhibitors prevented cytokine-mediated IκB-α degradation. Several cell-permeable ceramide analogs (C2, C6, and C8), hydrolysis products of sphingomyelin, activated NF-κB less potently than neutral SMase, but had no effect on NOx production. These results demonstrate an essential role of NF-κB activation in mediation of neutral SMase-induced iNOS expression, but distinct from the proteasome-mediated IκB-α degradation by cytokines, suggesting the possible involvement of an additional signaling pathway(s).
Potential Regulatory Roles for G Protein-Coupled Receptor Kinases and β-Arrestins in Gonadotropin-Releasing Hormone Receptor Signaling*
Authors: Neill Jimmy D. , Duck L. Wayne , Musgrove Lois C. , Sellers Jeffrey C. 
Published: January 2014

AbstractGnRH stimulates gonadotropin secretion, which desensitizes unless the releasing hormone is secreted or administered in a pulsatile fashion. The mechanism of desensitization is unknown, but as the GnRH receptor is G protein coupled, it might involve G protein-coupled receptor kinases (GRKs). Such kinases phosphorylate the intracellular regions of seven-transmembrane receptors, permitting β-arrestin to bind, which prevents the receptor from activating G proteins. Here, we tested the effect of GRKs and β-arrestins on GnRH-induced inositol trisphosphate (IP3) production in COS cells transfected with the GnRH receptor complementary DNA. GRK2, -3, and -6 overexpression inhibited IP3 production by 50–75% during the 30 sec of GnRH treatment. Coexpression of GRK2 and β-arrestin-2 suppressed GnRH-induced IP3 production more than that of either alone. Immunocytochemical staining of rat anterior pituitary revealed that all cells expressed GRK2, -3, and -6; all cells also expressed theβ -arrestins. Western blots on cytosolic extracts of rat pituitaries revealed the presence of GRK2/3 and β-arrestin-1 and -2. The expression of GRKs and β-arrestins by gonadotropes and their inhibition of GnRH-stimulated IP3 production in COS-1 cells expressing the GnRH receptor suggest a potential regulatory role for the GRK/β arrestin paradigm in GnRH receptor signaling.