bluuebunny/crossref_metadata_2025_split huggingface dataset:
Dynamic Changes in Prolactin Promoter Activation in Individual Living Lactotrophic Cells
Authors: Takasuka N. , White M. R. H. , Wood C. D. , Robertson W. R. , Davis J. R. E. 
Published: January 2014

The firefly luciferase gene has become widely used as a convenient reporter for studies of gene promoter regulation. Very recently, the development of ultralow-light imaging cameras has enabled the quantitative digital imaging of light signals resulting from luciferase activation in the presence of luciferin substrate. We have applied this technology to the study of PRL promoter activation in individual pituitary tumor cells to study the temporal and spatial characteristics of the expression of a well-characterized pituitary hormone gene.Rat pituitary GH3 cells were transfected by lipofection with a luciferase reporter gene linked to 5000 bp from the human PRL gene 5′-flanking region. A series of stably transfected cell clones were generated, and one of these was chosen for detailed study on the basis of appropriate regulation of high-level luciferase expression by a series of known stimuli including TRH, forskolin, the calcium channel agonist Bay K8644, and basic fibroblast growth factor (bFGF). These cells were subjected to direct imaging of luciferase activity using a Hamamatsu photon-counting camera linked to a Zeiss Axiovert microscope with an Argus-50 image processor. Cells were exposed to 1 mm luciferin, and images were integrated over 30-min periods for up to 72 h. The total photon count over a given field settled to steady levels within 10 h and then remained constant for over 55 h. Addition of forskolin, TRH, or bFGF increased the total photon count of fields of 20–100 cells by 2- to 4-fold consistent with previous data from transient expression assays using the human PRL promoter. Individual cells, on the other hand, showed marked marked temporal and spatial heterogeneity and variability of luciferase expression when studied at 3-h intervals. Unstimulated cells showed variable luciferase expression with up to 40-fold excursions in photon counts per single cell area within 12-h periods. Stimulation of cells with either TRH, forskolin, or bFGF resulted in smooth increases in photon output over fields of 20–100 cells, but again individual cell responses differed widely, with some cells showing slow progressive rises in photon output, others showing phasic or transient responses, and yet others showing no response.In conclusion, we found a surprising degree of heterogeneity and temporal variability in the level of gene expression in individual living pituitary tumor cells over long periods of time, with markedly divergent responses to hormonal or intracellular stimulation. The use of stably transfected clonal cell lines with extended periods of reporter gene imaging offers a valuable insight into control of gene expression in living cells in real time.
Reduced 11β-Hydroxysteroid Dehydrogenase Activity in the Remaining Kidney Following Nephrectomy*
Authors: Escher Geneviève , Vogt Bruno , Beck Thomas , Guntern Daniel , Frey Brigitte M. , Frey Felix J. 
Published: January 2014

AbstractIntracellular access of steroids to gluco- and mineralocorticoid receptors is regulated by reduced 11β-hydroxysteroid dehydrogenase (OHSD) 1 and 2. These enzymes convert active 11β-OH-steroids into inactive 11-keto-steroids. The purpose of the present study was to establish whether the 11β-OHSD1 and 11β-OHSD2 are modulated in the remnant kidney 24 h or 14 days after uninephrectomy (UNX) in rats. Overall, 11β-OHSD activity was analyzed by measuring the ratio of the exogenous 11β-OH-steroid prednisolone to its 11-keto metabolite prednisone in vivo in kidney tissue using high performance liquid chromatography. To determine which isoenzyme accounts for the changed activity 24 h after UNX, the oxidation and reduction attributable to 11β-OHSD1 and oxidation to 11β-OHSD2 were analyzed in total renal extracts and in isolated glomeruli, proximal convoluted tubules (PCT), cortical ascending limbs, and cortical convoluted tubules (CCT). The messenger RNA content of 11β-OHSD1 and 11β-OHSD2 was measured by RT-PCR in renal tissues and single segments, using glyceraldehyde-3-phosphate-dehydrogenase as an internal standard. Protein amounts of 11β-OHSD1 and 11β-OHSD2 were assessed by Western blot. The prednisolone/prednisone ratio increased 24 h after UNX in 9 out of 10 animals (P ≤ 0.0011), and was unchanged 14 days after UNX. 11β-OHSD1 oxidation (P ≤ 0.032) and reduction activity (P ≤ 0.002) declined 24 h after UNX in total extracts. 11β-OHSD1 oxidase activity was more than 3 times higher in PCT than in glomeruli, cortical ascending limbs, and CCT, and declined by 50% after UNX (P ≤ 0.001). The reductase activity did not change following UNX in PCT. 11β-OHSD2 activity was 5–15 times higher in CCT than in the other segments, and decreased significantly after UNX (P ≤ 0.008). UNX did not affect messenger RNA and protein levels of both enzymes in total renal extracts. In conclusion, 11β-OHSD1 and 11β-OHSD2 are predominantly expressed in PCT and CCT, respectively, and their corresponding oxidative activities decline after UNX. Thus, the access of 11β-glucocorticosteroids to gluco- and mineralocorticoid receptors in the remaining kidney is facilitated after UNX.
Growth Hormone and Mild Exercise in Combination Markedly Enhance Cortical Bone Formation and Strength in Old Rats*
Authors: Oxlund H. , Andersen N. B. , Ørtoft G. , Ørskov H. , Andreassen T. T. 
Published: January 2014

AbstractThe effects of a combination of mild exercise and GH injections on bone were studied in old female rats. Biosynthetic human GH, 2.7 mg/kg/day, was injected sc for 73 days. Exercised rats ran 8 m/min on a treadmill for 1 h/day. All rats (age 21 months old) were labeled with a tetracycline injection 56 days and a calcein injection 11 days before killing. The GH injections resulted in an 11-fold increase in femoral middiaphyseal bone formation rate and a 12% increase in cross-sectional area compared with the saline-injected group. The mild exercise doubled the mineralizing surface but did not influence the bone formation rate significantly. The combination of GH injections plus exercise, however, resulted in a further increase of 39% in bone formation rate, primarily at the anterolateral aspects, and an increase of 5% in cross-sectional area compared with the group injected with GH only. The femur ultimate breaking load was increased by 37% and the stiffness by 42% in the group injected with GH compared with the saline-injected group. Exercise alone did not influence the femur mechanical properties. The combination of GH injections plus exercise induced a 4% further increase in ultimate breaking load and 7% further increase in stiffness compared with the group injected with GH alone. The GH injections induced a 117% increase in serum insulin-like growth factor I. The GH-insulin-like growth factor I axis stimulates recruitment of osteoblast precursor cells, resulting in increased bone formation at the periosteal surface. GH injections and mild excercise in combination modulate and increase further the formation and strength of cortical bone in old female rats.
Molecular Cloning and Hormonal Regulation of a Murine Epididymal Retinoic Acid-Binding Protein Messenger Ribonucleic Acid
Authors: Lareyre Jean-Jacques , Zheng Weng-Li , Zhao Guang-Quan , Kasper Susan , Newcomer Marcia E. , Matusik Robert J. , Ong David E. , Orgebin-Crist Marie-Claire 
Published: January 2014

AbstractA complementary DNA encoding the mouse epididymal secretory protein MEP 10 (mouse epididymal protein 10) was cloned and is now renamed murine epididymal retinoic acid binding protein (mE-RABP). The analysis of the predicted primary amino acid sequence showed that mE-RABP has a 75% identity with rat ESP I (epididymal secretory protein I), another epididymal retinoic acid-binding protein. The homology strongly suggests that mE-RABP is the mouse orthologue of rat ESP I. A computer analysis of the predicted three-dimensional structure confirmed that mE-RABP can accommodate retinoic acid as ligand. In the rat, ESP I messenger RNA (mRNA) is expressed in the efferent ducts and in the entire caput epididymidis. However, in the mouse, the expression of a 950-bp mE-RABP mRNA was detected only in principal cells of the mid/distal caput epididymidis, suggesting that the regulation of region-specific expression is different in rat and mouse. Northern blot analyses showed that mE-RABP gene expression is no longer detected 10 days after castration but progressively rebounds between days 15 and 60. However, mE-RABP protein could not be detected by Western blot 30 days after castration. Androgen replacement, begun 5 days after castration and continued for 4 days restored significant expression of mE-RABP mRNA. Efferent duct ligation for 10 days did not affect gene expression. Taken together, these results indicate that mE-RABP mRNA expression is regulated by androgens but not by testicular factors. The overall similarity in the primary amino acid sequence of mE-RABP with ESP I and other members of the lipocalin superfamily suggests that they are evolutionarily related.
Pregnancy Increases Soluble and Particulate Guanylate Cyclases and Decreases the Clearance Receptor of Natriuretic Peptides in Ovine Uterine, But Not Systemic, Arteries*
Authors: Itoh Hiroaki , Bird Ian M. , Nakao Kazuwa , Magness Ronald R. 
Published: January 2014

AbstractPregnancy increases uterine blood flow by 30- to 50-fold and uterine production of cGMP by 38-fold. Moreover, cGMP causes potent vasodilatation. We hypothesized that pregnancy up-regulates soluble and particulate guanylate cyclases (sGC and pGC) in ovine uterine arteries. Activities of sGC and pGC were compared by measuring cGMP production (37 C; 10 min) by uterine arteries from nonpregnant (n = 5) and pregnant (n = 4, 120 ± 2 days’ gestation; term = 145 ± 3 days; mean ± se) ewes after sodium nitroprusside (100 μm), atrial natriuretic peptide (1μ m), or C-type natriuretic peptide (CNP; 1μ m) treatment. The protein and/or messenger RNA expressions of sGC β1-subunit, pGC-A, pGC-B, the clearance receptor of natriuretic peptide (CR), and CNP were investigated in uterine and systemic (renal and/or omental) arteries from nonpregnant (n = 29) and pregnant (n = 21; 125 ± 2 days’ gestation) ewes. The potencies of uterine arterial GC activities were sGC ≫ pGC-A&gt; pGC-B. Activities as well as protein expression of sGC, pGC-A, and pGC-B in pregnant uterine arteries were increased 48–128% above those in nonpregnant controls concomitant with a 34% down-regulation of CR protein expression; systemic arterial protein expressions were unaltered. These changes in uterine arterial GC-B and CR were confirmed using RT-PCR. Immunohistochemical staining of CNP in uterine, but not systemic, arterial endothelium from pregnant ewes was much stronger than that from nonpregnant ewes. Thus, two distinct GC pathways are present in ovine uterine artery, and both may be specifically up-regulated during pregnancy and so contribute to the tremendous local increase in cGMP production during pregnancy.
Phospholipase D- and Protein Kinase C Isoenzyme-Dependent Signal Transduction Pathways Activated by the Calcitonin Receptor*
Authors: Naro Fabio , Perez Marie , Migliaccio Silvia , Galson Deborah L. , Orcel Philippe , Teti Anna , Goldring Steven R. 
Published: January 2014

AbstractThe calcitonin receptor expressed by the porcine LLC-PK1 renal tubule cells is a seven-transmembrane domain, G protein-coupled receptor activating adenylyl-cyclase and phospholipase C. Salmon calcitonin stimulated dose- and time-dependent release of the phospholipase D-dependent phosphatidylcholine product[ 3H]choline with an EC50 = 2.5 ± 0.3 × 10−8m, similar to that determined for phosphoinositide metabolism (EC50 = 4.5 ± 1.0 × 10−8m). The hormone failed to induce release of [3H]phosphocholine and[ 3H]glycerophosphocholine, ruling out activation of phosphatydilcholine-specific phospholipase C and phospholipase A. Calcitonin stimulated phosphatidic acid, a product of phospholipase D-dependent phosphatydilcholine hydrolysis. Activation of phospholipase D was confirmed by release of [3H]phosphatydilethanol, a specific and stable product in the presence of a primary alcohol. Activation of calcitonin receptor induced diacylglycerol formation, with a rapid peak followed by a prolonged increase, due to activation of phospholipase C and of phospholipase D. Consequently, the protein kinase-Cα, but not the δ isoenzyme, was cytosol-to-membrane translocated by approximately 50% after 20 min exposure to calcitonin, whereas protein kinase-Cζ, which was approximately 40% membrane-linked in unstimulated cells, translocated by approximately 19%. The human calcitonin receptor expressed by BIN-67 ovary tumor cells, although displaying higher affinity for calcitonin, failed to activate phospholipase D and protein kinase-C in response to the hormone. This receptor lacks the G protein binding consensus site due to the presence of a 48-bp cassette encoding for a 16-amino acid insert in the predicted first intracellular loop. This modification is likely to prevent the calcitonin receptor from associating to phospholipase-coupled signaling.
Relaxin Increases the Accumulation of New Epithelial and Stromal Cells in the Rat Cervix during the Second Half of Pregnancy*
Authors: Burger Laura L. , Sherwood O. D. 
Published: January 2014

Abstract
Both cervical and vaginal growth are relaxin dependent during rat pregnancy. We recently reported a relaxin-dependent 1.5-fold increase in cervical and vaginal DNA content from midpregnancy until term. This finding indicated that relaxin probably promotes cervical and vaginal growth at least in part by promoting cellular proliferation. The objective of this study was to identify and quantify cells in the cervix and vagina that proliferate during the second half of rat pregnancy in response to relaxin.
Primiparous pregnant rats were ovariectomized or sham ovariectomized (group C; n = 8) on day 9 of pregnancy (D9). Ovariectomized rats were then treated with physiological doses of progesterone plus estrogen (n = 7) or progesterone, estrogen, and porcine relaxin (n = 7). Cellular proliferation was determined by continuously administering a low dose of 5-bromo-2′-deoxyuridine (BrdU) via miniature osmotic pump from D9–D22. On D22, cervices and vaginas were collected, fixed in formalin, paraffin embedded, and serially sectioned (4 μm). Adjacent serial sections were either immunostained for BrdU to assess cell proliferation or stained with hematoxylin to determine total cell number. Cell proliferation was evaluated by counting BrdU-positive nuclei and total nuclei in the same area on adjacent sections. Cell counts were determined using computerized digital morphometric analysis at ×575.
In control rats, nearly 75% of the epithelial cells and 55% of the stromal cells within the cervix at term had proliferated during the second half of pregnancy. The accumulation of approximately half of the new cells was relaxin dependent. Within the cervical stroma, relaxin increased the accumulation of cells associated with blood vessels and also the number of isolated cells (probably fibroblasts). Relaxin did not appear to affect smooth muscle cell proliferation in the cervix. In contrast to the cervix, a minority of vaginal epithelial cells (45%) and stromal cells (20%) proliferated during the second half of pregnancy. Although relaxin appeared to have a tendency to increase the accumulation of new vaginal epithelial and stromal cells, morphometric analysis did not provide support for such an effect.
In conclusion, this study demonstrates that relaxin promotes a marked increase in the accumulation of new epithelial cells and stromal cells within the cervix. The relaxin-induced increase in new epithelial and stromal cells probably contributes to relaxin’s effects on growth and remodeling of the cervix that are required for rapid and safe delivery.
Pregnancy-Specific Protein B Induces Release of an Alpha Chemokine in Bovine Endometrium
Authors: Austin Kathy J. , King Cathy P. , Vierk Judith E. , Sasser R. Garth , Hansen Thomas R. 
Published: January 2014

Abstract
Pregnancy-specific protein B (PSPB), is secreted from binucleate trophoblast of the bovine conceptus as early as day 15 of pregnancy. The objective of this experiment was to determine if PSPB induced uterine proteins. PSPB was purified from day 120 cotyledons using antibody-based affinity chromatography. Endometrium from day 14 non-pregnant cows (n = 3) was prepared for explant (3H-Leu added) culture. Radiolabeled proteins released into medium were dialyzed, separated using 1D-PAGE, and detected using fluorography and densitometry. PSPB (0, 0.5, 5, 25 & 50 nM) caused a concentration-dependent increase in the release of a radiolabeled 8-kDa uterine protein. Western blots revealed that the 8-kDa protein cross-reacted with antibody against granulocyte chemotactic protein-2 (GCP-2). PSPB also induced release of GCP-2 by bovine endometrial (BEND) cells in primary culture. The induction of GCP-2 by PSPB was blocked by addition of antiserum against PSPB (1:4 molar ratio). This is the first indication that PSPB has a hormonal role in inducing GCP-2, an alpha chemokine that also is induced by interferon-tau during early pregnancy. This chemotactic cytokine may be integral to mediating adhesion, inflammation and angiogenesis associated with early implantation.
The Estrogen Receptor β-Isoform (ERβ) of the Human Estrogen Receptor Modulates ERα Transcriptional Activity and Is a Key Regulator of the Cellular Response to Estrogens and Antiestrogens1
Authors: Hall Julie M. , McDonnell Donald P. 
Published: January 2014

Abstract
The human estrogen receptor α (ERα) and the recently identified ERβ share a high degree of amino acid homology; however, there are significant differences in regions of these receptors that would be expected to influence transcriptional activity. Consequently, we compared the mechanism(s) by which these receptors regulate target gene transcription, and evaluated the cellular consequences of coexpression of both ER subtypes. Previously, it has been determined that ERα contains two distinct activation domains, ERα-AF-1 and ERα-AF-2, whose transcriptional activity is influenced by cell and promoter context. We determined that ERβ, like ERα, contains a functional AF-2, however, the ERβ-AF-2 domain functions independently within the receptor. Of additional significance was the finding that ERβ does not contain a strong AF-1 within its amino-terminus but, rather, contains a repressor domain that when removed, increases the overall transcriptional activity of the receptor. The importance of these findings was revealed when it was determined that ERβ functions as a transdominant inhibitor of ERα transcriptional activity at subsaturating hormone levels and that ERβ decreases overall cellular sensitivity to estradiol. Additionally, the partial agonist activity of tamoxifen manifest through ERα in some contexts was completely abolished upon coexpression of ERβ. In probing the mechanisms underlying ERβ-mediated repression of ERα transcriptional activity we have determined that 1) ERα and ERβ can form heterodimers within target cells; and 2) ERβ interacts with target gene promoters in a ligand-independent manner. Cumulatively, these data indicate that one role of ERβ is to modulate ERα transcriptional activity, and thus the relative expression level of the two isoforms will be a key determinant of cellular responses to agonists and antagonists.
Losartan and Angiotensin II Inhibit Aldosterone Production in Anephric Rats via Different Actions on the Intraadrenal Renin-Angiotensin System*
Authors: Peters Jörg , Obermüller Nicholas , Woyth Alexander , Peters Barbara , Maser-Gluth Christiane , Kränzlin Bettina , Gretz Norbert 
Published: January 2014

Abstract
Angiotensin II (ANG II) is a major stimulator of aldosterone biosynthesis. When investigating the relative contribution of circulating and locally produced ANG II, we were therefore surprised to find that ANG II, given chronically sc (200 ng/kg·min), markedly inhibits a nephrectomy (NX)-induced rise of aldosterone concentrations (from 10 ± 2 to 465 ± 90 ng/100 ml in vehicle infused, and from 9 ± 2 to 177 ± 35 in ANG II infused rats 55 h after NX and hemodialysis). We further observed, by in situ hybridization, that bilateral NX increases the number of adrenocortical cells expressing renin and that this rise was prevented by ANG II. Moreover, the rise of aldosterone levels was also inhibited by the AT1-receptor antagonist, losartan (10 μg/kg·min, chronically ip from 8 ± 2 to 199 ± 26 ng/100 ml), despite the absence of circulating renin and a reduction of ANG I to less than 10%. These data demonstrate that aldosterone production, after NX, is regulated by an intraadrenal renin-angiotensin system and that this system is physiologically suppressed by circulating angiotensin.
Because the effects of losartan or ANG II on aldosterone production involved a latency period of at least 30 h after NX and were associated with a modulation or recruitment of renin-producing cells, we suggest that the intraadrenal renin-angiotensin system operates via regulation of cell differentiation on a long-term scale, rather than or additionally to its short-term effects on aldosterone synthase activity.
Differential Effects of Superior and Inferior Spermatic Nerves on Testosterone Secretion and Spermatic Blood Flow in Cats*
Authors: Chiocchio S. R. , Suburo A. M. , Vladucic E. , Zhu B. C. , Charreau E. , Décima E. E. , Tramezzani J. H. 
Published: January 2014

AbstractIt has been postulated that testosterone secretion is partially regulated by signals from the spermatic nerves. To further examine this hypothesis in vivo, the superior (SSN) or the inferior (ISN) spermatic nerves were stimulated electrically (varying intensity, 25 Hz, 0.2 msec, 10 min) in anesthetized cats, determining the testosterone concentration and the blood flow in the spermatic vein. In some additional experiments arterial blood was sampled, and norepinephrine (NE) output was calculated. Stimulation of the SSN (25–35 V) increased the testosterone concentration in spermatic vein blood (P &lt; 0.01 compared with prestimulation levels). The response varied among animals, reaching a 50–100% increase in some animals, whereas in others it ranged from almost undetectable to more than 10 ng/100 g·min. Under the same experimental conditions, the NE output increased from 135.4 ± 99 to 1614.2 ± 347 pg/ml (P &lt; 0.01), and spermatic blood flow decreased from 24.1 ± 1.42 to 20.2 ± 1.65 ml/min·100 g (P &lt; 0.05) during nerve stimulation. By contrast, stimulation of the ISN (25–35 V) modified neither the testosterone concentration, the NE output, nor the blood flow in the spermatic vein. High intensity stimulation (36–70 V) of each spermatic nerve evoked different vascular and hormonal effects. SSN activation induced a marked decrease in spermatic blood flow during stimulation and an increase in the testosterone response, whereas ISN activation resulted only in an enhanced spermatic blood flow. Our results suggest that testosterone secretion, although mainly dependent on gonadotropin secretion, could be further regulated by neural inputs from the SSN acting directly or alternatively through changes in blood flow. It would appear that the SSN mainly supplies the vasoconstrictor fibers to the testis, whereas the ISN provides vasodilator fibers.