bluuebunny/crossref_metadata_2025_split huggingface dataset:
Development of a Homologous Radioimmunoassay for Mouse Growth Hormone Receptor*
Authors: Camarillo Ignacio G. , Thordarson Gudmundur , Ilkbahar Yonca N. , Talamantes Frank 
Published: January 2014

Abstract
A RIA for mouse GH receptor (mGHR) was developed. A synthetic peptide corresponding to the carboxyl-terminal 14 amino acids of the mGHR (GHR-2 peptide) was used as the antigen for antiserum production. The synthetic peptide was also used as the standard and radioligand in the RIA. The ability of the antiserum to recognize the mGHR was demonstrated by quantitating receptor concentrations in liver and mammary gland from virgin and 15-day-pregnant mice. Serial dilutions of these samples yielded displacement curves parallel to the synthetic peptide. No significant cross-reactivity was seen with serum from virgin or 15-day-pregnant mice, mGH, recombinant mGH-binding protein (mGHBP), a synthetic peptide identical to the hydrophilic tail of mGHBP, or a 14-amino acid synthetic peptide corresponding to amino acids 338–351 of mGHR (GHR-1 peptide). The concentration range of the mGHR RIA was 0.5–200 nm, and the intra- and interassay coefficients of variation were 6.5% and 6.1%, respectively. The concentration of liver GHR increased significantly during pregnancy compared with that in virgin mice, from 0.246 ± 0.045 pmol/mg protein (mean ± sem; n = 5) in the virgin animals to 1.015 ± 0.159 pmol/mg protein (n = 5) in pregnant mice. In contrast, the mGHR concentration in the mammary gland decreased significantly during pregnancy from 0.606 ± 0.201 pmol/mg protein (mean ± sem; n = 5) to 0.299 ± 0.027 pmol/mg protein (n = 5). Comparison of the total number of binding sites in livers from virgin and pregnant mice using the GH RRA and the combined results of the mGHR and mGHBP RIAs showed that the two methods gave almost identical results for livers from virgin animals, or 0.363 ± 0.063 pmol/mg protein (mean± sem; n = 3) and 0.371 ± 0.008 pmol/mg protein (n = 3) for the GH RRA and the mGHR plus mGHBP RIAs, respectively. However, in livers from pregnant animals, the combined results from the mGHR and mGHBP RIAs were approximately 1.8 times higher than those obtained by the GH RRA, or 6.732 ± 0.612 pmol/mg protein (mean ± sem; n = 3) and 3.693 ± 0.67 pmol/mg protein (n = 3) for the mGHR plus the mGHBP RIAs and the GH RRA, respectively. The increase in the total GH binding capacity in livers from pregnant mice compared with those from virgin animals was largely due to an increase in the GHBP content. The increase in GHR was only 2.4-fold, or from 0.153 ± 0.01 pmol/mg protein (mean ± sem; n = 3) in virgin mice to 0.364 ± 0.03 pmol/mg protein (n = 3) in the 15-day-pregnant mice, whereas GHBP increased almost 30-fold during pregnancy, or from 0.218 ± 0.003 pmol/mg protein (mean ± sem; n = 3) in virgin animals to 6.369 ± 0.607 pmol/mg protein (n = 3) in pregnant mice.
Glucocorticoid-Induced Apoptosis and Regulation of NF-κB Activity in Human Leukemic T Cells*
Authors: Ramdas Jyoti , Harmon Jeffrey M. 
Published: January 2014

AbstractGlucocorticoid-induced apoptosis was investigated in glucocorticoid-sensitive 6TG1.1 and resistant ICR27TK.3 human leukemic T cells. Following glucocorticoid treatment of 6TG1.1 cells, chromatin fragmentation was observed after a delay of 24 h. Fragmentation was not observed in ICR27TK.3 cells containing mutant glucocorticoid receptors (L753F) that are activation-deficient but retain the ability to repress AP-1 activity. Nor was fragmentation observed after treatment with RU38486, indicating that repression of AP-1 activity is not involved. As described in other systems, fragmentation required ongoing protein synthesis. However, inhibition of protein synthesis with cycloheximide anytime during the first 18 h of steroid treatment was as effective in blocking chromatin fragmentation as inhibition for the entire period, suggesting that synthesis of a component with a rapid turnover rate is required. Dexamethasone treatment completely blocked 12-O-tetradecanoylphorbol 13-acetate induction of nuclear factor-κB (NF-κB) activity and elicited an increase in the amount of immunoreactive IκBα in sensitive 6TG1.1 cells but not in resistant ICR27TK.3 cells. In addition, mild detergent treatment of cell extracts indicated that a substantial amount of cytoplasmic NF-κB is complexed with IκBα or some other inhibitory factor. These results suggest that induction of a labile inhibitory factor such as IκBα may contribute to glucocorticoid-induced apoptosis.
Pituitary Lactotroph Adenomas Develop after Prolonged Lactotroph Hyperplasia in Dopamine D2 Receptor-Deficient Mice1
Authors: Asa Sylvia L. , Kelly Michele A. , Grandy David K. , Low Malcolm J. 
Published: January 2014

AbstractTuberoinfundibular dopamine tonically inhibits PRL expression and secretion from the pituitary gland by the activation of dopamine D2 receptors (D2R) localized on lactotrophs. Mutant female mice that lack D2Rs have persistent hyperprolactinemia but also develop extensive hyperplasia of pituitary lactotrophs and peliosis of the adenohypophysis at 9 to 12 months of age, while age-matched male D2R-deficient mice have no morphologic adenohypophysial lesion. We now report that both female and male D2R-deficient mice 17 to 20 months of age develop pituitary lactotroph adenomas. Of 12 aged female mice examined, all developed monohormonal PRL-immunoreactive neoplasms that had a characteristic juxtanuclear Golgi pattern of PRL staining and loss of the reticulin fiber network. Several of these adenomas were 50-fold larger than normal glands with marked suprasellar extension and invasion of brain but no gross evidence of distant metastases. They also had striking peliosis that was more marked than the lesion seen in the hyperplastic pituitaries of the younger females. These findings demonstrate that a chronic loss of neurohormonal dopamine inhibition promotes the hyperplasia-neoplasia sequence in adenohypophysial lactotrophs. Our results are analogous to previous data indicating that protracted stimulation of adenohypophysial cells by hormones or growth factors results in proliferation with initial hyperplasia followed by the development of neoplasia. Six aged male D2R-deficient mice had slightly enlarged anterior pituitaries similar in size to normal female glands. However, each case exhibited multifocal, microscopic lactotroph adenomas with strong nuclear immunoreactivity for estrogen receptors and Pit-1 transcription factor. The unexpected development of adenomas in males without preexisting or concomitant hyperplasia suggests that prolonged loss of dopamine inhibition may also cause neoplasia by distinct cellular mechanisms in male and female animals.
Pituitary Adenylate Cyclase-Activating Polypeptide Is an Auto/Paracrine Stimulator of Acute Progesterone Accumulation and Subsequent Luteinization in Cultured Periovulatory Granulosa/Lutein Cells*
Authors: Gräs Søren , Hannibal Jens , Fahrenkrug Jan 
Published: January 2014

Abstract
Recently, we have demonstrated that pituitary adenylate cyclase-activating polypeptide (PACAP) is transiently expressed in steroidogenic ovarian cells during the periovulatory period. This prompted us to establish an in vitro system in which the potential local regulatory role of PACAP during periovulatory progesterone production could be examined. Granulosa/lutein cells from PMSG- and human CG (hCG)-stimulated immature rats were used. The cells were isolated from preovulatory follicles 4–6 h after the hCG injection, at which time the transient ovarian PACAP expression begins in vivo. By immunocytochemistry on intact cells and RIA on cell extracts and culture medium, granulosa/lutein cells were found to accumulate and secrete PACAP during incubation. Furthermore, the cells responded to exogenous PACAP 38 with a rapid (10−7m induced a peak value 20-fold higher than controls at 2 h) and dose-dependent accumulation of progesterone. PACAP 38 (5 × 10−9m), in combination with an approximately half-maximal dose of hCG (1 ng/ml), showed an additive effect on progesterone accumulation. Immunoneutralization of endogenously released PACAP was performed using the IgG fraction from a specific PACAP antiserum that dose-dependently inhibits the progesterone accumulating effect of exogenous PACAP 38. The acute effects of endogenously released PACAP were studied during 8 h of incubation of granulosa/lutein cells with anti-PACAP IgG (100 μg/ml). A significant reduction in progesterone accumulation was observed after 4, 6, and 8 h [38.7% (P &lt; 0.05), 41.2% (P &lt; 0.02), and 50% (P &lt; 0.002), respectively], compared with nonimmune IgG (100 μg/ml) treated cultures. The long-term effects on luteinization induced by endogenously released PACAP were studied after incubation of the cells with anti-PACAP IgG or nonimmune IgG for 24 h, followed by incubation for 9 days in serum-containing medium. Under these conditions, nonimmune IgG-treated cells assumed a luteal phenotype, accumulating large and stable amounts of progesterone and acquiring hypertrophic cell bodies with numerous lipid droplets and distinct nucleoli in the large nuclei. Anti-PACAP IgG-treated cells displayed morphological and functional signs of impaired luteinization being smaller and more irregular and with progesterone accumulation being significantly lower throughout the incubation period [56.4% (P &lt; 0.02), 69.2% (P &lt; 0.05), 43.8% (P &lt; 0.02), and 52.2% (P &lt; 0.02) at 1, 4, 7, and 10 days, respectively]. Together, these findings support an auto- or paracrine role for PACAP during gonadotropin-induced acute periovulatory progesterone production and subsequent luteinization in granulosa/lutein cells.
A Naturally Occurring Genetic Variant in the Human Chorionic Gonadotropin-β Gene 5 Is Assembly Inefficient*
Authors: Miller-Lindholm Amanda K. , Bedows Elliott , Bartels Cynthia F. , Ramey Jacques , Maclin Victoria , Ruddon Raymond W. 
Published: January 2014

AbstractThe hCGβ gene family is composed of six homologous genes linked in tandem repeat on chromosome 19; the order of the genes is 7, 8, 5, 1, 2, and 3. Previous studies have shown that hCGβ gene 5 is highly expressed during the first trimester of pregnancy. The purpose of our study was to identify naturally occurring polymorphisms in hCGβ gene 5 and determine whether these alterations affected hCG function. The data presented here show that hCGβ gene 5 was highly conserved in the 334 asymptomatic individuals and 41 infertile patients examined for polymorphisms using PCR followed by single stranded conformational polymorphism analysis. Most of the polymorphisms detected were either silent or located in intron regions. However, one genetic variant identified in β gene 5 exon 3 was a G to A transition that changed the naturally occurring valine residue to methionine in codon 79 (V79M) in 4.2% of the random population studied. The V79M polymorphism was always linked to a silent C to T transition in codon 82 (tyrosine). To determine whether βV79M hCG had biological properties that differed from those of wild-type hCG, a β-subunit containing the V79M substitution was created by site-directed mutagenesis and was coexpressed with the glycoprotein hormone α-subunit in Chinese hamster ovary cells and 293T cells. When we examined βV79M hCG biosynthesis, we detected atypical βV79M hCG folding intermediates, including a βV79M conformational variant that resulted in aβ -subunit with impaired ability to assemble with the α-subunit. The inefficient assembly of βV79M hCG appeared to be independent ofβ -subunit glycosylation or of the cell type studied, but, rather, was due to the inability of the βV79M subunit to fold correctly. The majority of the V79M β-subunit synthesized was secreted as unassembled free β. Although the amount of αβ hCG heterodimer formed and secreted by βV79M-producing cells was less than that by wild-type β-producing cells, the hCG that was secreted as αβ V79M heterodimer exhibited biological activity indistinguishable from that of wild-type hCG.
Parathyroid Hormone-Related Protein in Rat Penis: Expression, Localization, and Effect on Cavernosal Pressure1
Authors: Lang Hervé , Endlich Nicole , Lindner Véronique , Endlich Karlhans , Massfelder Thierry , Stewart Andrew F. , Saussine Christian , Helwig Jean-Jacques 
Published: January 2014

Abstract
Although PTH-related protein-(1–36) [PTHrP-(1–36)] is known to be expressed in smooth muscle and to exert potent myorelaxant effects, its tonic effects on cavernosal smooth muscle has not yet been explored. Using the RT-PCR technique, the present study establishes that PTHrP messenger RNA is present in microdissected corpus cavernosa in the rat. In immunohistochemical studies using affinity-purified antibodies to middle regions of PTHrP, immunostaining was localized throughout the penile structures, including vessels, cavernosal smooth muscle, and trabecular fibroblasts. Strong immunostaining for PTHrP was also detected in the dorsal nerve bundles. In anesthetized rats, intracavernosally injected boluses of increasing doses of PTHrP-(1–36) (0.3–30 pmol in 100 μl saline) had little effect on intracavernosal pressure. However, they markedly potentiated the dilatory response to papaverine (8–800 nmol), increasing the papaverine-induced intracavernous pressure by 2.5-fold, close to the mean arterial pressure. In conclusion, the cavernosal expression of PTHrP messenger RNA, the distribution of immunoreactive PTHrP throughout the structuro-functional components of the erectile apparatus and its strong potentiating action on papaverine-induced cavernosal relaxation, collectively suggest that PTHrP participates in the control of cavernosal tone.
In Situ Estrogen Synthesized by Aromatase P450 in Uterine Leiomyoma Cells Promotes Cell Growth Probably Via an Autocrine/Intracrine Mechanism*
Authors: Sumitani Hiroshi , Shozu Makio , Segawa Tomoya , Murakami Kouichi , Yang Hui-Juan , Shimada Keiko , Inoue Masaki 
Published: January 2014

Abstract
In the present study we characterized in detail the expression of aromatase P450 in leiomyomas to determine the role of in situ estrogen in the growth advantage of leiomyomas. The levels of aromatase P450 transcripts were determined by quantitative RT-PCR to be significantly higher in leiomyomas than in corresponding myometrium. The overexpression of aromatase P450 in leiomyomas was also confirmed by Western blot analysis. The estimated size of immunoreactive aromatase was 58 kDa, similar to that in placenta. To identify a cell type that express aromatase P450 in leiomyomas, histological specimens were stained for aromatase P450 using a polyclonal antibody. Strong immunoreactivity was detected in the cytoplasm of leiomyoma cells, whereas surrounding normal myometrium displayed weak or negative staining. Smooth muscle-like cells in culture obtained from leiomyomas, positive for actin D fiber, possessed immunoreactive granules of aromatase in the cytoplasm. Conversion of androgen to estrogen was effectively stimulated by phorbol myristate acetate and dexamethasone plus interleukin-1β and was completely abolished by selective inhibitors of aromatase P450 (fadrozole and TZA-2209), but not by inhibitors of 5α-reductase (finasteride and flutamide). The apparent Km of androstenedione was 3 nm in the presence of dexamethasone and interleukin-1β, corresponding to the plasma concentration of androstenedione in women of reproductive age. To determine whether endogenous aromatase P450 plays a role in the growth promotion of leiomyoma cells, we evaluated the cell growth of smooth muscle-like cells treated with various concentrations of estrogen and androgen using a WST-1 assay. Treatment with testosterone (10−8 and 10−7m) and androstenedione (10−8 and 10−7m) stimulated the growth of smooth muscle-like cells obtained from leiomyomas to the same extent as estradiol (10−10–10−7m), whereas dihydrotestosterone (10−11–10−8m) did not. The stimulatory effect of testosterone on cell growth was again abolished by cotreatment with fadrozole. The level of estradiol in the medium of testosterone (10−8m)-treated smooth muscle-like cells was 10−11m, which was 1 order lower than the minimum concentration of estradiol necessary to promote cell growth (10−10m). This indicates that estradiol synthesized in leiomyomas promotes their growth via an autocrine/intracrine mechanism. We conclude that myometrial cells of leiomyomas overexpress aromatase P450 and are able to synthesize sufficient estrogen to accelerate their own cell growth. Overexpression of aromatase P450 may play a role in the growth advantage of leiomyoma tissue over surrounding myometrium via an autocrine/intracrine mechanism.
Multiple Cadherin Superfamily Members with Unique Expression Profiles Are Produced in Rat Testis1
Authors: Johnson Kamin J. , Patel Sutchin R. , Boekelheide Kim 
Published: January 2014

AbstractAdhesion between germ and Sertoli cells is thought to be crucial for spermatogenesis. Cadherin superfamily proteins, including classic cadherins and protocadherins, are important mediators of cell-cell adhesion. Using a degenerate PCR cloning strategy, we surveyed the expression of cadherin superfamily members in rat testis. Similar to brain, testis expressed a large number of cadherin superfamily members: 7 classic cadherins of both types I and II, 14 protocadherins, 2 protocadherin-related cadherins, and 1 cadherin-related receptor-like protein. All three protocadherin families (α, β, and γ) were found in testis. Using a semiquantitative RT-PCR assay, messenger RNA expression was determined for each cadherin superfamily member during a postnatal developmental time-course and following ablation of specific testis cell types by ethanedimethanesulfonate, methoxyacetic acid, and 2,5-hexanedione. Diverse expression patterns were observed among the cadherins, suggesting that cadherin expression is cell type-specific in testis. The large number and variety of cadherin superfamily members found in testis supports a critical function for cadherin-mediated cell-cell adhesion in spermatogenesis.
Influence of the in Vivo Calcium Status on Cellular Calcium Homeostasis and the Level of the Calcium-Binding Protein Calreticulin in Rat Hepatocytes*
Authors: Mailhot Geneviève , Petit Jean-Luc , Demers Christian , Gascon-Barré Marielle 
Published: January 2014

AbstractLittle attention has been given to the consequences of the in vivo calcium status on intracellular calcium homeostasis despite several pathological states induced by perturbations of the in vivo calcium balance. The aim of these studies was to probe the influence of an in vivo calcium deficiency on the resting cytoplasmic Ca2+ concentration and the inositol-1,4,5-trisphosphate-sensitive Ca2+ pools. Studies were conducted in hepatocytes (a cell type well characterized for its cellular Ca2+ response) isolated from normal and calcium-deficient rats secondary to vitamin D depletion. Both resting cytoplasmic Ca2+ concentration and Ca2+ mobilization from inositol-1,4,5-trisphosphate -sensitive cellular pools were significantly lowered by calcium depletion. In addition, Ca deficiency was shown to significantly reduce calreticulin messenger RNA and protein levels but calcium entry through store-operated calcium channels remained unaffected, indicating that the Ca2+ entry mechanisms are still fully operational in calcium deficiency. The effects of calcium deficiency on cellular calcium homeostasis were reversible by repletion with oral calcium feeding alone or by the administration of the calcium-regulating hormone 1,25-dihydroxyvitamin D3, further strengthening the tight link between extra- and intracellular calcium. These data, therefore, challenge the currently prevailing hypothesis that extracellular Ca2+ has no significant impact on cellular Ca2+ by demonstrating that despite the large Ca2+ gradient between extra- and intracellular Ca2+ concentrations, calcium deficiency in vivo significantly alters the hormone-sensitive cellular calcium homeostasis.
Expression of Estrogen Receptor α and β in the Rhesus Monkey Corpus Luteum during the Menstrual Cycle: Regulation by Luteinizing Hormone and Progesterone*
Authors: Duffy Diane M. , Chaffin Charles L. , Stouffer Richard L. 
Published: January 2014

Abstract
There are conflicting reports on the presence or absence of estrogen receptor (ER) in the primate corpus luteum, and the discovery of a second type of estrogen receptor, ERβ, adds an additional level of complexity. To reevaluate ER expression in the primate luteal tissue, we used semiquantitative RT-PCR based assays and Western blotting to assess ERα and β messenger RNA (mRNA) and protein levels in corpora lutea (n = 3/stage) obtained from adult female rhesus monkeys at early (days 3–5), mid (days 6–8), mid-late (days 10–12), and late (days 14–16) luteal phase of the natural menstrual cycle. ERα mRNA levels did not vary across the stages of the luteal phase, and ERα protein was not consistently detected in luteal tissues. However, ERβ mRNA and protein levels were detectable in early and mid luteal phases and increased (P &lt; 0.05) to peak levels at mid-late luteal phase before declining by late luteal phase. To determine if ERβ mRNA expression in the corpus luteum is regulated by LH, monkeys received the GnRH antagonist antide either alone or with 3 daily injections of LH to simulate pulsatile LH release. Treatment with antide alone or concomitant LH administration did not alter luteal ERβ mRNA levels. When monkeys also received the 3β-hydroxysteroid dehydrogenase inhibitor trilostane to reduce luteal progesterone production, luteal ERβ mRNA levels were 3-fold higher (P &lt; 0.05) than in monkeys receiving antide + LH only. Replacement of progestin activity with R5020 reduced luteal ERβ mRNA levels to those seen in animals receiving antide + LH. Thus, there is dynamic ERβ expression in the primate corpus luteum during the menstrual cycle, consistent with a role for estrogen in the regulation of primate luteal function and life span via a receptor (ERβ)-mediated pathway. Increased ERβ expression in the progestin-depleted corpus luteum during LH exposure suggests that the relative progestin deprivation experienced by the corpus luteum between LH pulses may enhance luteal sensitivity to estrogens during the late luteal phase of the menstrual cycle.
Dissociation of Locomotor Activation and Suppression of Food Intake Induced by CRF in CRFR1-Deficient Mice
Authors: Contarino Angelo , Dellu Françoise , Koob George F. , Smith George W. , Lee Kuofen , Vale Wylie W. , Gold Lisa H. 
Published: January 2014

ABSTRACT
Corticotropin-releasing factor (CRF) systems are involved in locomotor and feeding behaviors. Two distinct CRF receptor subtypes, CRFR1 and CRFR2, are thought to mediate CRF actions in the central nervous system. However, the role for each receptor in locomotor activity and feeding remains to be determined. Using CRFR1 null mutant mice, the present study examined the functional significance of this receptor in ambulation and feeding. CRF treatment of wild-type mice resulted in increased levels of locomotion whereas no change was observed in CRFR1-deficient mice as compared to vehicle-treated mutant mice. In contrast, CRF decreased food-water intake in both wild type and CRFR1-deficient mice equally. These results support an important role for CRFR1 in mediating CRF-induced locomotor activation, whereas other receptor subtypes, likely CRFR2, may mediate the appetite-suppressing effects of CRF-like peptides.