bluuebunny/crossref_metadata_2025 huggingface dataset:
Regulation of phosphatidylinositol-phosphate kinase IIgamma gene transcription by thyroid-stimulating hormone in thyroid cells
Authors: Park S , Lee W , You KH , Kim H , Suh JM , Chung HK , Shong M , Kwon OY
Published: December 2004

This study was performed to evaluate the effects of thyroid-stimulating hormone (TSH) on phosphatidylinositol-4-phosphate 5-kinase type IIgamma (PIPKIIgamma) gene expression in the thyrocytes of FRTL-5 cells. Although PIPKIIgamma mRNA was expressed constantly in the absence of added TSH, its expression increased remarkably in the presence of 10(-9) M TSH. This increase started within 6 h of the addition of TSH, and reached a maximum at 8 h. The mRNA expression properties of PIPKIIgamma in the cells were identified using inhibitors. Actinomycin D blocked PIPKIIgamma transcription strongly, while cycloheximide did not. In an experiment using 5,6-dichlo-1-beta-d -ribofuranosylbenzimidaxole, the half-life of PIPKIIgamma mRNA was approximately 6 h in the presence or absence of TSH, and it was not affected by the stability of the PIPKIIgamma mRNA. The effects of TSH on PIPKIIgamma gene expression were specific, and other growth factors examined (transferrin, insulin and hydrocortisone) did not alter its expression. It is possible that the mechanism of PIPKIIgamma gene expression is involved in the permissive effect of the TSH-cAMP cascade proper. Our results indicate, for the first time, that the expression of PIPKIIgamma is regulated transcriptionally by TSH in thyrocytes.
Expression and role of functional glucocorticoid receptors in the human androgen-independent prostate cancer cell line, DU145
Authors: Dondi D , Maggi R , Scaccianoce E , Martini L , Motta M , Poletti A
Published: December 2004

We investigated the presence of glucocorticoid receptors (GR) as well as the role of glucocorticoids (Gc) in the control of proliferation of the androgen-independent prostate cancer cell line, DU145. We detected the presence of a specific high affinity binding site (K(d) 2.3 nM) for [(3)H]dexamethasone ([(3)H]Dex) in the cytosolic preparations of DU145 cells; the density of these binding sites is significantly higher than that detected in HA22T/VGH and in HepG2, two hepatoma cell lines classically considered models for the study of GR. Immunocytochemistry studies confirmed the presence of GR in the cytosolic compartment of DU145 cells; GR undergo translocation to the nucleus following exposure to dexamethasone (Dex). The functional activity of GR present in DU145 cells was also studied by analyzing the potency of Dex in inducing chloramphenicol acyltransferase (CAT) activity in DU145 cells transfected with a glucocorticoid/progesterone response element (GRE/PRE) tkCAT plasmid (GRE/PREtkCAT plasmid). The results have shown that Dex stimulates the transcriptional activity of GR in transfected DU145 cells with an EC(50) of 9.65 nM and a maximal induction of sevenfold above basal levels. Finally, a dose-dependent (IC(50) 3.14 nM) decrease of DU145 cell numbers was observed after their exposure to Dex for 4 days; this effect was counteracted by the presence of the steroid antagonist, RU486. In conclusion, the present data suggest a possible role of corticoids in the control of the growth of androgen-independent prostate cancer.
Developmental and hormonal regulation of type II DNA topoisomerase in rat testis
Authors: Bakshi RP , Galande S , Bali P , Dighe R , Muniyappa K
Published: December 2004

Type II DNA topoisomerase (topo II) is required for diverse biological functions including DNA replication, maintenance of genome stability, chromosome segregation and chromosome condensation. While the identity of topo II in rodent testis has been established, the regulation of topo II expression during the development of the postnatal testis and gametogenesis is unclear. Here, we report that rat testis topo II is developmentally and hormonally regulated. Topo IIalpha mRNA levels peaked prior to the onset of puberty, declined sharply thereafter and stabilized in adult testis. In contrast, the topo II enzyme content was lower in prepubertal testis but increased after the onset of puberty. Topo II was expressed in a cell-specific manner within germ cells, being detected only in pachytene spermatocytes. While testosterone markedly increased topo IIalpha mRNA levels in prepubertal testis, continued treatment failed to enhance topo IIalpha mRNA above postpubertal control levels. The extent of topo II activity remained steady regardless of the testosterone-induced increase in topo IIalpha mRNA levels. Inhibition of testosterone function in postpubertal animals by ethanedimethane sulphonate (EDS) and flutamide resulted in a significant decrease in topo IIalpha gene expression and topo II activity. The administration of exogenous testosterone (T) to EDS- and flutamide-treated rats restored topo IIalpha mRNA levels and topo II activity similar to the levels seen in the testis of age-matched control animals. Histochemical analyses of testes indicated that the effect of T on spermatogenesis was separable from its effect on topo IIalpha expression. Our results reveal that testosterone acts as a positive regulator of topo IIalpha gene expression and is required for the maintenance of topo IIalpha expression during the development of the postnatal testis and spermatogenesis.
The carboxy-terminal domain of IGF-binding protein-5 inhibits heparin binding to a site in the central domain
Authors: Song H , Shand JH , Beattie J , Flint DJ , Allan GJ
Published: December 2004

The IGF-binding protein (IGFBP)-5 protein contains consensus heparin binding motifs in both its carboxy (C)-terminal and central domains, although only the C-terminal site has previously been shown to be functional. We have made two chimeric IGFBP proteins by switching domains between rat IGFBP-5 and -2, named BP552 and BP522 to reflect the domains present, and a truncated rat IGFBP-5 mutant (1-168), named BP550. The ability of these proteins and wild-type (wt) IGFBPs-5 and -2 to bind to either IGFs or heparin was determined using biosensor real-time analysis and heparin ligand blotting respectively. We report that the chimeric molecules have IGF binding affinities comparable to those of the native IGFBPs from which they were derived and, as expected, the binding of BP550 to IGFs was greatly compromised. More surprising was the finding that the ability of BP552 and BP550 to bind to heparin was equivalent to that of wtIGFBP-5, whereas wtIGFBP-2 and BP522 failed to bind. These results demonstrate that the active heparin binding site in BP552 and BP550 is contained within the central domain of IGFBP-5, and that this site is active only in the absence of the C-terminal domain. We subsequently mutated two basic amino acids (R136A:R137A) in the central consensus binding sites between residues 132-140. This resulted in the loss of heparin binding for BP550, confirming the importance of these two basic amino acids in the central domain heparin binding activity. In light of these findings, we suggest that C-terminally truncated fragments of IGFBP-5 generated in vivo by proteolysis could retain heparin/extracellular matrix binding properties.
The transfection-induced overexpression of IGF-binding protein-4 affects the secretory activity of porcine ovarian granulosa cells and their response to hormones and IGF-I
Authors: Sirotkin AV , Makarevich AV , Corkins MR , Kotwica J , Bulla J
Published: December 2004

The aim of our studies was to examine whether IGF-binding protein (IGFBP)-4 is involved in the control of the secretion of various ovarian substances and also the mediation of the effects of several hormones and growth factors on this secretion. For this purpose, we carried out the transfection of porcine granulosa cells with a cDNA sense construct, increasing IGFBP-4 synthesis. We then compared the release of IGFBP-3, progesterone, oxytocin and IGF-I by control and transfected cells cultured with and without porcine LH (100 ng/ml), porcine GH (100 ng/ml), IGF-I (10 ng/ml), oxytocin (10 ng/ml) and estradiol-17beta (100 ng/ml). The concentration of IGFBP-4 produced was assessed using ligand blotting, and the release of progesterone, oxytocin, IGF-I and IGFBP-3 was evaluated using RIA/IRMA techniques. It was observed that GH, IGF-I, estradiol, LH and oxytocin alter the progesterone, oxytocin, IGF-I and IGFBP-3 release by porcine ovarian granulosa cells. Transfection of these cells with an IBFBP-4 cDNA expression construct significantly increased the IGFBP-4 accumulation in cell-conditioned medium. Furthermore, this transfection significantly reduced progesterone, oxytocin and IGFBP-3 release, and increased IGF-I output in cells cultured in the absence or presence of GH, IGF-I, estradiol and LH. The addition of oxytocin, but not of other tested substances, fully or partially prevented the effects of IGFBP-4 overexpression on IGFBP-3, IGF-I, but not on progesterone release. The present results suggested that IGFBP-4, as well as GH, IGF-I, estradiol, LH and oxytocin, is a potent regulator of porcine ovarian steroid (progesterone), nonapeptide hormone (oxytocin), growth factor (IGF-I) and growth factor-binding protein (IGFBP-3) release. IGFBP-4 is an inhibitor of basal progesterone, oxytocin and IGFBP-3 release and a stimulator of IGF-I output by porcine ovarian cells. The action of IGFBP-4 on the ovary can be mediated by (1) inhibition of oxytocin release, (2) suppression of receptor/postreceptor events induced by other hormones and IGF-I and (3) stimulation of IGF-I release.
Molecular evolution of GH in primates: characterisation of the GH genes from slow loris and marmoset defines an episode of rapid evolutionary change
Authors: Wallis OC , Zhang YP , Wallis M
Published: December 2004

Pituitary growth hormone (GH), like several other protein hormones, shows an unusual episodic pattern of molecular evolution in which sustained bursts of rapid change are imposed on long periods of very slow evolution (near-stasis). A marked period of rapid change occurred in the evolution of GH in primates or a primate ancestor, and gave rise to the species specificity that is characteristic of human GH. We have defined more precisely the position of this burst by cloning and sequencing the GH genes for a prosimian, the slow loris (Nycticebus pygmaeus) and a New World monkey, marmoset (Callithrix jacchus). Slow loris GH is very similar in sequence to pig GH, demonstrating that the period of rapid change occurred during primate evolution, after the separation of lines leading to prosimians and higher primates. The putative marmoset GH is similar in sequence to human GH, demonstrating that the accelerated evolution occurred before divergence of New World monkeys and Old World monkeys/apes. The burst of change was confined largely to coding sequence for mature GH, and is not marked in other components of the gene sequence including signal peptide, 5' upstream region and introns. A number of factors support the idea that this episode of rapid change was due to positive adaptive selection. Thus (1) there is no apparent loss of function of GH in man compared with non-primates, (2) after the episode of rapid change the rate of evolution fell towards the slow basal level that is seen for most mammalian GHs, (3) the accelerated rate of substitution for the exons of the GH gene significantly exceeds that for introns, and (4) the amino acids contributing to the hydrophobic core of GH are strongly conserved when higher primate and other GH sequences are compared, and for coding sequences other than that coding for hydrophobic core residues the rate of substitution for non-synonymous sites (K(A)) is significantly greater than that for synonymous sites (K(S)). In slow loris, as in most non-primate mammals, there is no evidence for duplication of the GH gene, but in marmoset, as in rhesus monkey and man, the putative GH gene is one of a cluster of closely related genes.
Alternative splicing of the proadrenomedullin gene results in differential expression of gene products
Authors: Martinez A , Hodge DL , Garayoa M , Young HA , Cuttitta F
Published: December 2004

The adrenomedullin (AM) gene codifies for two bioactive peptides, AM and proAM N-terminal 20 peptide (PAMP). We have found two forms of the AM mRNA. Form A is devoid of introns and results in a prohormone containing both peptides. Form B retains the third intron, which introduces a premature stop codon, producing a shorter prohormone with only PAMP. Tissues with a higher B/A ratio were more immunoreactive for PAMP than for AM. The form B message was found in the cytoplasmic compartment, thus excluding that the longer message was a result of contaminating nuclear mRNA. Form B was found in cells that express PAMP but not AM. mRNA expression in a variety of cell lines was investigated by ribonuclease protection assay and form B was found in significant amounts in two of them. Treatments that modify AM expression, such as exposure to hypoxia, were shown to change the B/A ratio and the relative secretion of AM and PAMP, indicating that the splicing mechanism for AM can be modulated and is physiologically relevant. Analysis of the sequence of the third intron and the fourth exon of the AM gene found motifs compatible with a highly regulated alternative splicing mechanism.
The engineered, cytosolic form of human type I 3beta-hydroxysteroid dehydrogenase/isomerase: purification, characterization and crystallization
Authors: Thomas JL , Mason JI , Blanco G , Veisaga ML
Published: December 2004

Human type I 3beta-hydroxysteroid dehydrogenase/isomerase (3beta-HSD/isomerase) is an integral membrane protein of human placental trophoblast and of insect Sf9 cells transfected with recombinant baculovirus containing the cDNA encoding the enzyme. Purified native or wild-type enzyme remains in solution only in the presence of detergent that may prevent crystallization. The membrane-spanning domain (residues 283-310) of the enzyme protein was deleted in the cDNA using PCR-based mutagenesis. The modified enzyme was expressed by baculovirus in the cytosol instead of in the microsomes and mitochondria of the Sf9 cells. The cytosolic form of 3beta-HSD/isomerase was purified using affinity chromatography with Cibacron Blue 1000. The NAD(+) and NaCl used to elute the enzyme were removed by size-exclusion centrifugation. Hydroxylapatite chromatography yielded a 26-fold purification of the enzyme. SDS-PAGE revealed a single protein band for the purified cytosolic enzyme (monomeric molecular mass 38.8 kDa) that migrated just below the wild-type enzyme (monomeric molecular mass 42.0 kDa). Michaelis-Menten constants measured for 3beta-HSD substrate (dehydroepiandrosterone) utilization by the purified cytosolic enzyme (K(m)=4.5 microM, V(max)=53 nmol/min per mg) and the pure wild-type enzyme (K(m)=3.7 microM, V(max)=43 nmol/min per mg), for isomerase substrate (5-androstene-3,17-dione) conversion by the purified cytosolic (K(m)=25 microM, V(max)=576 nmol/min per mg) and wild-type (K(m)=28 microM, V(max)=598 nmol/min per mg) enzymes, and for NAD(+) reduction by the 3beta-HSD activities of the cytosolic (K(m)=35 microM, V(max)=51 nmol/min per mg) and wild-type (K(m)=34 microM, V(max)=46 nmol/min per mg) enzymes are nearly identical. The isomerase activity of the cytosolic enzyme requires allosteric activation by NADH (K(m)=4.6 microM, V(max)=538 nmol/min per mg) just like the wild-type enzyme (K(m)=4.6 microM, V(max)=536 nmol/min per mg). Crystals of the purified, cytosolic enzyme protein have been obtained. The inability to crystallize the detergent-solubilized, wild-type microsomal enzyme has been overcome by engineering a cytosolic form of this protein. Determining the tertiary structure of 3beta-HSD/isomerase will clarify the mechanistic roles of potentially critical amino acids (His(261), Tyr(253)) that have been identified in the primary structure.
Cross-talk between Stat5b and estrogen receptor-alpha and -beta in mammary epithelial cells
Authors: Bjornstrom L , Kilic E , Norman M , Parker MG , Sjoberg M
Published: December 2004

Both 17beta-estradiol and prolactin play important roles in the mammary gland, raising the possibility of functional cross-talk between the two signaling pathways. Here, we demonstrate that estrogen receptor-alpha (ERalpha) and -beta (ERbeta) are both able to potentiate transcription from a Stat5-responsive promoter when activated by prolactin. Potentiation was observed not only in the presence of 17beta-estradiol, but also in the presence of anti-estrogens such as tamoxifen and ICI 182,780. The magnitude of the response was dependent on cell-type: in the HC11 mouse mammary epithelial cell line ERbeta potentiates transcription efficiently whereas ERalpha showed low activity. Conversely, in COS-7 cells, both estrogen receptors were active. We show that activation domains in the N-terminus (AF-1) and the C-terminus (AF-2) of the ERs are dispensable for potentiation. The effects are dependent on the presence of an intact DNA-binding/hinge domain, which we show is capable of interacting with Stat5b in vitro and in HC11 cell extracts. We conclude that ERalpha and ERbeta act as coactivators for Stat5b through a mechanism which is independent of AF-1 and AF-2.
Nuclear post-transcriptional control of gene expression
Authors: Akker SA , Smith PJ , Chew SL
Published: December 2004

The mammalian nucleus has considerable control over nascent transcripts. The basic mechanisms of post-transcriptional processing are well understood and recently some of the principles underlying the regulation of nuclear processing events have been elucidated. Here we review the recent progress in identification of signalling pathways that modulate the action of key RNA-binding proteins which regulate splicing, and the mechanisms of action of the C-terminal domain of RNA polymerase II that co-ordinate transcription with nuclear mRNA processing events.
Oxytocin receptor gene expression in rat mammary gland: structural characterization and regulation
Authors: Breton C , Di Scala-Guenot D , Zingg HH
Published: December 2004

The differential, tissue-specific regulation of oxytocin (OT) binding sites allows the neurohypophysial nonapeptide OT to fulfill a dual role: to induce uterine contractions at parturition and to mediate milk ejection during lactation. Whereas uterine OT binding sites are up-regulated prior to parturition and are rapidly down-regulated thereafter, mammary gland OT binding sites gradually increase throughout gestation and remain up-regulated during the ensuing lactation period. Here, we structurally characterized OT receptor (OTR) mRNA in mammary gland and analyzed its expression during gestation and lactation and in response to steroid treatment. In mammary gland tissues, we found a 6.7 and a 5.4 kb OTR mRNA species, and both species were further analyzed by RACE (rapid amplification of cDNA ends). The 6.7 kb mRNA was found to be common to mammary gland and uterus and to extend 618 nucleotides beyond the published sequence of the rat OTR gene. The 5.4 kb mRNA species is unique to the mammary gland and terminates at a mammary gland-specific polyadenylation site that is not preceded by a classical polyadenylation signal. RT-PCR analysis did not provide any evidence for differences in the coding regions, suggesting that both uterine and mammary gland OTR mRNAs encode the same receptor protein. Furthermore, primer extension experiments showed that no differences exist in the specific transcriptional initiation sites of the OTR gene in the two tissues. During pregnancy, OTR mRNA per mammary gland increased approximately 150-fold and remained high during lactation, consistent with the previously identified regulation of OT binding sites and the role of OT during lactation. Whereas estrogen administration strongly induced the uterine OTR mRNA levels (>5-fold), mammary gland remained unaffected by steroid treatment. Moreover, tamoxifen had no effect on the mammary gland OTR mRNA level. In summary, our data demonstrate a differential control of OTR expression in uterus versus mammary gland and a mammary gland-specific OTR mRNA polyadenylation site. However, this differential control apparently does not involve the expression of different receptor genes nor the utilization of tissue-specific transcriptional initiation sites.
Purification and molecular characterization of recombinant rat betacellulin
Authors: Dunbar AJ , Priebe IK , Sanderson MP , Goddard C
Published: December 2004

A method for the large scale expression and purification of rat betacellulin (BTC) from Escherichia coli has been developed using a cleavable fusion protein strategy. Insoluble fusion protein collected as inclusion bodies was dissolved in urea under reducing conditions, re-folded, and purified by gel filtration chromatography and C(4) RP-HPLC. Authentic rat BTC was obtained after proteolytic cleavage of the fusion protein with Factor Xa. Factor Xa cleaved an additional site within the BTC protein, generating a truncated isoform separable from full-length BTC by heparin-affinity chromatography. Recombinant rat BTC stimulated the proliferation of mouse Balb/c 3T3 fibroblasts and competed for binding to the ErbB1 receptor in a dose-dependent manner analogous to that of BTC purified from natural sources.
Liver receptor homologue-1 is expressed in the adrenal and can regulate transcription of 11 beta-hydroxylase
Authors: Wang ZN , Bassett M , Rainey WE
Published: December 2004

Liver receptor homologue-1 (LRH-1, designated NR5A2) is a mammalian homologue of Drosophila fushi tarazu factor (dFTZ-F1) and structurally belongs to the orphan nuclear receptor superfamily. LRH-1 can recognize the DNA sequence 5'-AAGGTCA-3', the canonical recognition motif for steroidogenic factor 1 (SF-1). Herein, we hypothesized that LRH-1 might play a role in the regulation of human adrenal expression of steroidogenic enzymes. To test this hypothesis, LRH-1 expression in human adult and fetal adrenal glands was examined by RT-PCR analysis. The fetal and adult adrenal glands, as well as liver and pancreas, were observed to express LRH-1 mRNA using RT-PCR. The ability of LRH-1 to enhance transcription of the gene encoding human 11 beta- hydroxylase (hCYP11B1) was then examined using the H295R adrenal cell line. LRH-1 co-transfection with hCYP11B1 luciferase promoter constructs caused a 25-fold induction of luciferase activity. Furthermore, co-transfection of a hCYP11B1 reporter construct containing a mutation in the SF-1 binding cis-element abolished the stimulatory effect of both SF-1 and LRH-1. Electrophoretic mobility shift assay (EMSA) demonstrated that LRH-1 could bind to the SF-1 response element. Taken together, our data suggested that LRH-1 is expressed in the adrenal, and can substitute for SF-1 to enhance transcription of genes encoding certain of the steroid-metabolizing enzymes. A role for LRH-1 in the regulation of adrenal or gonadal steroid hormone production should be further studied.
Regulation of cell cycle and cyclins by 16alpha-hydroxyestrone in MCF-7 breast cancer cells
Authors: Lewis JS , Thomas TJ , Klinge CM , Gallo MA , Thomas T
Published: December 2004

It has been suggested that alterations in estradiol (E(2)) metabolism, resulting in increased production of 16alpha-hydroxyestrone (16alpha-OHE(1)), is associated with an increased risk of breast cancer. In the present study, we examined the effects of 16alpha-OHE(1)on DNA synthesis, cell cycle progression, and the expression of cell cycle regulatory genes in MCF-7 breast cancer cells. G(1) synchronized cells were treated with 1 to 25 nM 16alpha-OHE(1) for 24 and 48 h. [(3)H]Thymidine incorporation assay showed that 16alpha-OHE(1) caused an 8-fold increase in DNA synthesis compared with that of control cells, whereas E(2) caused a 4-fold increase. Flow cytometric analysis of cell cycle progression also demonstrated the potency of 16alpha-OHE(1) in stimulating cell growth. When G(1) synchronized cells were treated with 10 nM 16alpha-OHE(1) for 24 h, 62+/-3% of cells were in S phase compared with 14+/-3% and 52+/-2% of cells in the control and E(2)-treated groups respectively. In order to explore the role of 16alpha-OHE(1) in cell cycle regulation, we examined its effects on cyclins (D1, E, A, B1), cyclin dependent kinases (Cdk4, Cdk2), and retinoblastoma protein (pRB) using Western and Northern blot analysis. Treatment of cells with 10 nM 16alpha-OHE(1) resulted in 4- and 3-fold increases in cyclin D1 and cyclin A, respectively, at the protein level. There was also a significant increase in pRB phosphorylation and Cdk2 activation. In addition, transient transfection assay using an estrogen response element-driven luciferase reporter vector showed a 15-fold increase in estrogen receptor-mediated transactivation compared with control. These results show that 16alpha-OHE(1) is a potent estrogen capable of accelerating cell cycle kinetics and stimulating the expression of cell cycle regulatory proteins.
The putative androgen receptor-A form results from in vitro proteolysis
Authors: Gregory CW , He B , Wilson EM
Published: December 2004

Activation domains in the 114 kDa androgen receptor (AR) NH(2)- and carboxyl-terminal regions are thought to contribute to different extents to AR-mediated transactivation. We investigated using anti-peptide antibodies whether smaller AR forms that migrate like the previously described 87 kDa AR-A occur in vivo resulting in constitutive or increased gene activation. Immunoblots of prostate cancer and fibroblast cell culture extracts revealed 114 and 84 kDa AR forms. Antibody mapping indicated the 84 kDa AR lacked the ligand-binding domain and comigrated with the constitutively active AR fragment AR1-660. AR expressed in COS cells was 114 and 92 kDa. Migration of the 92 kDa AR was slightly slower than that of a 90 kDa expressed fragment that was designed to initiate at the second methionine (residue 189) and lacked the NH(2)-terminal FxxLF interaction sequence. The 92 kDa AR did not result from alternative initiation since it was observed when the second methionine was changed to alanine. Optimization of extraction conditions indicated that both 84 and 92 kDa forms resulted from in vitro proteolytic cleavage and that cleavage by caspase-3 could account for the 92 kDa form. The results suggest that AR forms with gel mobility similar to that of the previously described 87 kDa AR-A result from in vitro proteolytic cleavage of NH(2)- or carboxyl-terminal regions during cell extraction and storage and that smaller forms with increased transcriptional activity do not occur in vivo.
Secretory activity of bovine ovarian granulosa cells transfected with sense and antisense insulin-like growth factor (IGF) binding protein-3 and the response to IGF-I, GH, LH, oxytocin and oestradiol
Authors: Sirotkin AV , Makarevich AV , Corkins MR , Kotwica J , Kwon HB , Bulla J , Hetenyi L
Published: December 2004

The aim of our in vitro experiments was to examine if IGF binding protein (IGFBP)-3 is involved in control of bovine ovarian secretory activity. For this purpose we performed the transfection of bovine granulosa cells with cDNA sense and antisense constructs increasing or inhibiting IGFBP-3 synthesis. The release of IGFBP-3, progesterone, oxytocin, IGF-I and prostaglandins F (PGF) and E (PGE) by control and transfected cells was compared. The transfected ovarian cells were cultured with and without bLH (100 ng/ml), bGH (100 ng/ml), IGF-I (10 ng/ml), oxytocin (10 ng/ml) and oestradiol-17beta (100 ng/ml). The concentration of IGFBP-3 produced was assessed using ligand and western blotting and secretion of progesterone, oxytocin, IGF-I, PGF and PGE was evaluated using RIA/IRMA techniques. Transfection of cells with the sense IGFBP-3 cDNA construct resulted in the expected increase in IGFBP-3 release, whereas the antisense IGFBP-3 construct induced the expected reduction in IGFBP-3 output. The granulosa cells transfected to overexpress IGFBP-3 had an increase in IGF-I, PGF and PGE release, and a decrease in basal and hormone- or growth factor-induced accumulation of progesterone and oxytocin. The granulosa cells transfected to have reduced IGFBP-3 expression gave primarily significant opposite findings. The present results suggest the involvement of IGFBP-3 in control of bovine ovarian steroid, peptide hormone, growth factor and prostaglandin release. IGFBP-3 is a physiological stimulator of IGF-I and prostaglandin release and an inhibitor of steroid and peptide hormone output.
Molecular cloning of the cDNAs for pituitary glycoprotein hormone alpha subunits of two species of duck and their gene regulation
Authors: Hsieh YL , Chatterjee A , Chien JT , Yu JY
Published: December 2004

The cDNAs encoding pituitary glycoprotein hormone alpha subunits (PGHalphas) of two species of duck (Muscovy duck, Cairina moschata and Pekin duck, Anas platyrhynchos domesticus) were cloned and sequenced to better understand the phylogenic diversity and evolution of PGHalpha molecules in vertebrates. Oligonucleotide primers were designed and used for reverse transcription PCR (RT-PCR) amplification of PGHalpha cDNA fragments from total cellular RNA of pituitary glands. The remaining sequences were completed by rapid amplification of the cDNA ends. The nucleotide sequence of isolated PGHalpha cDNA of both ducks are identical, including 81 bp of 5' untranslated region (UTR), 360 bp of coding region, and 272 bp of 3'-UTR followed by a 13 bp poly(A)(+) tract. The total number of amino acids deduced from the cDNA of the duck PGHalpha is 120 with a signal peptide of 24 amino acids and a mature protein of 96 amino acids. PGHalphas of the ducks (order Anseriformes) share 96% homology of amino acid sequence in signal peptide, and 100% homology in mature proteins with chicken, quail and turkey (order Galliformes). Our data thus demonstrate identical inter-order homology of PGHalpha mature protein in birds. Ten cysteine residues, presumably forming five disulfide bonds within the alpha subunit, and four proline residues, presumably responsible for folding of the molecule, are conserved in the alpha subunit of ducks. Northern blot analysis revealed that PGHalpha mRNA is expressed only in the pituitary. In order to study factors regulating the gene expression of PGHalpha mRNA, duck pituitary fragments were incubated with GnRH, TRH, testosterone, or triiodothyronine (T(3)). GnRH and TRH increased, while testosterone and T(3) decreased, PGHalpha mRNA levels. This is the first report in birds of TRH up-regulation and down-regulation by testosterone and T(3) under in vitro conditions. The present study demonstrates both ducks have the same cDNA nucleotide and deduced amino acid sequences in the PGHalpha subunit, exhibiting identical inter-genus homology within the family of Anatidae. The findings from mRNA expression work suggest that hypothalamic GnRH and TRH up-regulate, while testosterone and T(3) down-regulate, PGHalpha gene expression in ducks.
Expression profile of active genes in the human pituitary gland
Authors: Tanaka S , Tatsumi K , Okubo K , Itoh K , Kawamoto S , Matsubara K , Amino N
Published: December 2004

To characterize transcripts abundantly expressed in the human pituitary gland in general as well as to isolate novel transcripts expressed specifically in this gland, we generated an expression profile of the active genes transcribed in it. A total of 1015 randomly collected 3prime prime or minute expressed sequence tags (ESTs) (gene signatures, GSs) were grouped into 527GS species. The results showed the relative expression levels of genes in the pituitary gland. The genes comprising more than 1% of total mRNA were prolactin, growth hormone and chromogranin B genes. When known genes were categorized, the genes for pituitary hormones were the most actively transcribed, followed by the genes for ribosomal proteins, nuclear proteins and secretory granule proteins. Through comparison of this gene expression profile with the BodyMap database containing profiles generated from 63 other human tissues, we obtained 11 genes which appeared to be specifically expressed in the pituitary gland. In addition to the eight known genes, we identified three novel pituitary-specific transcripts which encode putative proteins: pituitary gland specific factor 1a (PGSF1a), PGSF1b and PGSF2. This expression profile method is a novel approach to the isolation of pituitary-specific genes that may have important functions.
Negative regulation of nuclear factor-kappaB activation and function by glucocorticoids
Authors: Almawi WY , Melemedjian OK
Published: December 2004

Glucocorticoids (GCs) exert their anti-inflammatory and antiproliferative effects principally by inhibiting the expression of cytokines and adhesion molecules. Mechanistically, GCs diffuse through the cell membrane, and bind to their inactive cytosolic receptors (GRs), which then undergo conformational modifications that allow for their nuclear translocation. In the nucleus, activated GRs modulate transcriptional events by directly associating with DNA elements, compatible with the GCs response elements (GRE) motif, and located in variable copy numbers and at variable distances from the TATA box, in the promoter region of GC-responsive genes. In addition, activated GRs also acted by antagonizing the activity of transcription factors, in particular nuclear factor-kappaB (NF-kappaB), by direct and indirect mechanisms. GCs induced gene transcription and protein synthesis of the NF-kappaB inhibitor, IkappaB. Activated GR also antagonized NF-kappaB activity through protein-protein interaction involving direct complexing with, and inhibition of, NF-kappaB binding to DNA (Simple Model), or association with NF-kappaB bound to the kappaB DNA site (Composite Model). In addition, and according to the Transmodulation Model, GRE-bound GR may interact with and inhibit the activity of kappaB-bound NF-kappaB via a mechanism involving cross-talk between the two transcription factors. Lastly, GR may compete with NF-kappaB for nuclear coactivators, including CREB binding protein and p300, thereby reducing and inhibiting transcriptional activation by NF-kappaB. It should be noted that, in exerting its effect, activated GR did not affect the correct assembly of the pre-initiation (DAB) complex, but acted rather more proximally in inhibiting the correct assembly of transcription factors in the promoter region, and thus transcriptional initiation.
Prolonged dietary treatment with conjugated linoleic acid stimulates porcine muscle peroxisome proliferator activated receptor gamma and glutamine-fructose aminotransferase gene expression in vivo
Authors: Meadus WJ , MacInnis R , Dugan ME
Published: December 2004

Peroxisome proliferator activated receptors (PPARs) represent a family of DNA binding proteins that are activated by a variety of dietary and endogenous fatty acids. The PPAR proteins are expressed throughout the body and are the target of a variety of lipidaemic and insulin sensitizing drugs. Conjugated linoleic acid (CLA) is a collective name for octadecadienoic acid isomers with conjugated double bonds, which can also act as ligands for some of the PPAR family. To gain better understanding of the long-term effects of PPAR activation, CLA was fed at 11 g/kg of feed for 45 days to castrated male pigs (barrows). These barrows had a significant repartitioning of subcutaneous fat to lean tissue in the carcass: fat was reduced by 9 x 2% and lean muscle was increased by 3 x 5%, but intramuscular fat content was also increased by 14% (P<0 x 05). PPARgamma, glutamine-fructose aminotransferase (GFAT), adipocyte fatty acid binding protein (AFABP), but not PPARalpha mRNA levels were significantly increased (P<0 x 05) in the CLA-fed pigs. The increased expression of PPARgamma and AFABP indicates that CLA induced the development of preadipocytes from stromal-vascular (s-v) stem cells to promote intramuscular fat content. The increase in the expression of GFAT mRNA indicates that the glucose supply of the muscle cells had been increased with the CLA diet, possibly sparing intramuscular fatty acid reserves.
Hepatocyte growth factor stimulates proliferation of pancreatic beta-cells particularly in the presence of subphysiological glucose concentrations
Authors: Gahr S , Merger M , Bollheimer LC , Hammerschmied CG , Scholmerich J , Hugl SR
Published: December 2004

We investigated the role of hepatocyte growth factor (HGF) in beta-cell growth and its complex intracellular signal transduction pathways. Cell proliferation was measured in the beta-cell line INS-1 using [3H]thymidine incorporation. Activation of mitogenic signaling proteins was assessed using co-immunoprecipitation, immunoblot analysis and specific protein activity inhibitors in proliferation assays. HGF (1 x 375 nM) increased INS-1 cell proliferation in the presence of 3-24 mM glucose up to 45-fold vs unstimulated controls. HGF exceeded the effect of glucose alone (2 x 2-fold at 3 mM glucose and 1 x 7-fold in the presence of 15 mM glucose). The HGF-induced INS-1 cell proliferation was further increased by addition of IGF-I or GH. Stimulation with HGF activated the JAK-2/STAT-5 pathway with a subsequent activation of phosphatidylinositol-3'-kinase (PI3'K). PI3'K activation was necessary for HGF- and glucose-stimulated INS-1 cell proliferation. The effect of PI3'K was mediated via 70 kDa S6 kinase and protein kinase B, which showed maximum activation in the presence of 3-6 mM glucose. Protein kinase C was essential for HGF-induced INS-1 cell proliferation. The HGF effect was also mediated at low glucose concentrations via insulin receptor substrate 4 (IRS-4) whereas other IRS proteins did not show any activation. High glucose concentrations also showed an increased IRS-4/PI3'K binding and therefore activation. In conclusion, beta-cell proliferation is mediated via complex interacting signal transduction pathways. HGF, in contrast to other growth factors, seems to be of importance particularly in the presence of low glucose concentrations and therefore takes a special role in this complex concert.
Characterization of oestrogen receptors in zebrafish (Danio rerio)
Authors: Bardet PL , Horard B , Robinson-Rechavi M , Laudet V , Vanacker JM
Published: December 2004

We cloned the cDNAs corresponding to three oestrogen receptors (ERs) in zebrafish (Danio rerio). Sequence analysis and phylogenetic studies demonstrated that two of these genes, ER beta.1 and ER beta.2, arose from duplication of the original ER beta in many species of the fish phylum, whereas ER alpha is unique. Zebrafish ERs behaved as oestrogen-dependent transcription factors in transactivation assays. However, their reactivity to various oestrogen modulators was different compared with that of mouse ERs. ER mRNA expression during zebrafish development is restricted to distinct time periods, as observed by RNase protection assays. ER beta.2 is initially expressed as maternally transmitted RNA, until 6 h after fertilization, when expression disappears. Between 6 and 48 h after fertilization, no ER expression could be observed. After 48 h after fertilization, all ERs, but predominantly ER alpha, began to be expressed. We conclude that oestrogen signal transduction can operate during zebrafish development only within discrete time windows.
Vasopressin V2 receptor antagonists
Authors: Verbalis JG
Published: December 2004

Hyponatremia, whether due to the syndrome of inappropriate antidiuretic hormone secretion (SIADH) or disorders of water retention such as congestive heart failure and cirrhosis, is a very common problem encountered in the care of medical patients. To date, available treatment modalities for disorders of excess arginine vasopressin (AVP) secretion or action have been limited and suboptimal. The recent discovery and development of nonpeptide AVP V(2) receptor antagonists represents a promising new treatment option to directly antagonize the effects of elevated plasma AVP concentrations at the level of the renal collecting ducts. By decreasing the water permeability of renal collecting tubules, excretion of retained water is promoted, thereby normalizing or improving hypo-osmolar hyponatremia. In this review, SIADH and other water retaining disorders are briefly discussed, after which the published preclinical and clinical studies of several nonpeptide AVP V(2) receptor antagonists are summarized. The likely therapeutic indications and potential complications of these compounds are also described.
An Ets motif in the proximal decidual prolactin promoter is essential for basal gene expression
Authors: Brar AK , Kessler CA , Handwerger S
Published: December 2004

Transcriptional regulation of the prolactin gene in the decidua differs from that in the pituitary. Several lines of evidence strongly suggest this difference is due to regulation of the prolactin gene in the decidua and other extra-pituitary tissues by tissue-specific transcription factors, which activate distinct promoters to induce prolactin gene expression in extra-pituitary sites compared with the pituitary. The human decidua is a major site of extra-pituitary expression of the prolactin gene. Here we present evidence that the transcription factor Ets-1 is critical for basal expression of the decidua-type (or decidual) prolactin promoter. Overexpression of Ets-1 significantly induces decidual prolactin promoter activity in BeWo and JAR cells that express little or no endogenous Ets-1. Conversely, a dominant/negative mutant of Ets represses basal promoter activity. Although the proximal 1.5 kb of the decidual prolactin promoter contains six Ets motifs, only the Ets motif at nt -77/-71 is essential for basal gene expression. Mutation of the Ets motif at nt -77/-71 results in an approximately 90% decrease in promoter activity, while mutation of the other Ets motifs results in only small changes. Electrophoretic mobility shift assays demonstrate that Ets proteins in decidualized endometrial stromal cells bind this Ets motif in the decidual prolactin promoter. Ets protein expression increases up to 20-fold upon induction of decidualization in endometrial stromal cells under conditions in which expression of the prolactin gene is also induced. These studies provide strong evidence for a critical role of the Ets transcription factor in basal expression of the decidual prolactin promoter.
Biochemical origins of the non-monotonic receptor-mediated dose-response
Authors: Kohn MC , Melnick RL
Published: December 2004

A mathematical model was created to examine how xenobiotic ligands that bind to nuclear receptor proteins may affect transcriptional activation of hormone-regulated genes. The model included binding of the natural ligand (e.g. hormone) and xenobiotic ligands to the receptor, binding of the liganded receptor to receptor-specific DNA response sequences, binding of co-activator or co-repressor proteins (Rp) to the resulting complex, and the consequent transcriptional rate relative to that in the absence of the xenobiotic agent. The model predicted that the xenobiotic could act as a pure agonist, a pure antagonist, or a mixed agonist whose dose-response curve exhibits a local maximum. The response to the agent depends on the affinity of the liganded receptor-DNA complex for binding additional transcription factors (e.g. co-activator proteins). An inverted U-shaped dose-response occurred when basal levels of the natural ligand did not saturate receptor binding sites and the affinity for co-activator is weaker when the xenobiotic ligand is bound to the receptor than when the endogenous ligand is bound. The dose-response curve shape was not dependent on the affinity of the receptor for the xenobiotic agent; alteration of this value merely shifted the curve along the concentration axis. The amount of receptor, the density of DNA response sequences, and the affinity of the DNA-bound receptor for Rp determine the amplitude of the computed response with little overall change in curve shape. This model indicates that a non-monotonic dose-response is a plausible outcome for xenobiotic agents that activate nuclear receptors in the same manner as natural ligands.
Estradiol up-regulates estrogen receptor messenger ribonucleic acid in endometrial carcinoma (Ishikawa) cells by stabilizing the message
Authors: Robertson JA , Farnell Y , Lindahl LS , Ing NH
Published: December 2004

Estrogens up-regulate expression of the estrogen receptor alpha (ER) gene in most mammalian tissues studied. Using the ovariectomized ewe as a model, we determined that estradiol (E(2)) acted post-transcriptionally to increase endometrial ER mRNA concentrations by enhancing the stability of the message. The purpose of this study was to determine whether a similar E(2) effect occurs in Ishikawa cells, a well-differentiated human endometrial adenocarcinoma cell line. The presence and function of ER protein in Ishikawa cells was demonstrated by transactivation of a transfected plasmid (ERE(2)tkCAT) in response to 10(-)(9) M E(2), resulting in a 550% increase in reporter gene RNA. Ishikawa cells also responded to E(2) by up-regulating their ER mRNA concentration an average of 100% between 7 and 24 h of treatment. The effect of E(2) on ER mRNA stability was measured after blocking transcription with actinomycin D to find that the half-life increased from 6 to 10 h in control and E(2)-treated cells respectively. These results are consistent with cell-free studies which showed significant enhancement of the half-life of radiolabeled ER 3' untranslated region (3'UTR) RNA in extracts from E(2)-treated cells versus those from control cells. Thus, Ishikawa cells provide a relevant model system for the study of E(2)-regulated endometrial gene expression.
Response element sequence modulates estrogen receptor alpha and beta affinity and activity
Authors: Kulakosky PC , McCarty MA , Jernigan SC , Risinger KE , Klinge CM
Published: December 2004

The relationship between estrogen receptor (ER)-estrogen response element (ERE) binding affinity and estradiol (E(2))-induced transcription has not been systematically or quantitatively tested. We examined the influence of ERE palindrome length and the 3' ERE flanking sequence on ERalpha and ERbeta affinity binding in vitro and on the induction of reporter gene activity in transfected cells. The addition of one nucleotide in each arm of the 13 bp ERE palindrome, forming a 15 bp ERE palindrome, increased ERalpha and ERbeta affinity and transcription. In contrast, the addition of an AT-rich flanking sequence from genes highly stimulated by E(2) had little effect on affinity or reporter gene activity. Notable differences between ERalpha and ERbeta include: both K(d) and transcriptional induction were generally higher for ERalpha than ERbeta, better correlation between ERE palindrome length and transcriptional induction for ERalpha than ERbeta, and a better correlation between (ER-ERE)K(d) and transcriptional induction for ERalpha than for ERbeta.