bluuebunny/crossref_metadata_2025 huggingface dataset:
Calcitonin: characterisation and expression in a teleost fish, Fugu rubripes
Authors: Clark MS , Bendell L , Power DM , Warner S , Elgar G , Ingleton PM
Published: December 2004

The present report describes the structure and expression of the calcitonin gene in Fugu rubripes. It is composed of 4 exons and 3 introns. Splicing of exons 1, 2 and 3 generates the calcitonin pre-proprotein, while splicing of exons 1, 2 and 4 generates calcitonin gene-related protein (CGRP). Exons 1 and 2 encoding the signal sequence and the N-terminal peptide are common in both the gene products and this gene organisation has been conserved in human, rat, chicken and salmon. The gene environment around calcitonin in Fugu has been poorly conserved when compared with human, apart from a small gene cluster. The calcitonin gene in Fugu has a widespread tissue distribution but it is most highly expressed in the brain. The abundance of gene expression in the ultimobranchial gland and the pituitary indicates that these are important sites of production and that the peptide is probably secreted into the circulation and/or acts as a paracrine or autocrine controlling factor. Whilst the function of calcitonin in fish is still largely unknown, the distribution described here suggests that one of the potential functions may be as a neuropeptide.
Chicken neuropeptide Y-family receptor Y4: a receptor with equal affinity for pancreatic polypeptide, neuropeptide Y and peptide YY
Authors: Lundell I , Boswell T , Larhammar D
Published: December 2004

Within the neuropeptide Y (NPY) family of peptides, pancreatic polypeptide is the most divergent across species. It differs in 20 of 36 positions between human and chicken. In mammals, it binds primarily to the Y4 receptor, to which NPY and peptide YY (PYY) bind with lower affinities. Because of these large sequence differences in pancreatic polypeptide, we decided to characterise the chicken Y4 receptor. We report here that Y4 displays the least sequence conservation among the Y-family receptors, with only 57-60% overall amino acid identity between chicken and mammals, compared with 64-83% for the Y1, Y2 and Y5 receptors. After expression of the chicken Y4 receptor in COS-7 cells, (125)I-labelled porcine (p) PYY bound with a K(d) of 20 pM. In competition with (125)I-pPYY, chicken pancreatic polypeptide bound with high affinity at 140 pM. Interestingly, chicken PYY bound with even greater affinity at 68 pM. The affinity of NPY, 160 pM, was similar to that of pancreatic polypeptide. Chicken Y4 is less sensitive than is mammalian Y4 to truncation of the amino terminus of the NPY molecule. RT-PCR revealed expression in several peripheral organs, including adipose tissue and oviduct. In brain, Y4 mRNA was detected in the brainstem, cerebellum and hippocampus. In situ hybridisation to brain sections showed expression in the dorsal motor nucleus of the vagus in the brainstem. Thus the chicken Y4 receptor is less selective and anatomically more widespread than that in mammals, probably reflecting the original properties of the Y4 receptor.
RXR acts as a coregulator in the regulation of genes of the hypothalamo-pituitary axis by thyroid hormone receptors
Authors: Laflamme L , Hamann G , Messier N , Maltais S , M-F Langlois
Published: December 2004

Thyroid hormone receptors (TRs) often modulate transcriptional activity of target genes by heterodimerization with the 9-cis retinoic acid receptor (RXR). On positive thyroid response elements (TREs), RXR favors binding of the TR-RXR complex to DNA and stimulates transcription. RXR action on negative TREs is unclear. Furthermore, the single half-site configuration of many negative TREs does not favor the binding of a classic TR-RXR heterodimer. In a comparative study using CV-1 cells (relatively RXR- and TR-deficient) and JEG-3 cells (relatively TR-deficient), we demonstrate the importance of RXR in the negative transcriptional regulation of genes of the hypothalamo-pituitary axis by tri-iodothyronine. While RXR has variable effects on ligand-independent activation produced by TRs, it was required for efficient ligand-dependent repression of the TRH gene for TRalpha1 and TRbeta1 and of the TSH genes by all TRs. Using different RXR constructs we also observed the importance of the C-terminus of RXR but not of the N-terminus nor the DNA-binding domain, in the potentiation of negative regulation. We thus suggest that, with regard to negative regulation of the TRH and TSH genes by thyroid hormones, RXR behaves more like a cofactor than a classic heterodimerization partner.
Development of cDNA microarray for expression profiling of estrogen-responsive genes
Authors: Inoue A , Yoshida N , Omoto Y , Oguchi S , Yamori T , Kiyama R , Hayashi S
Published: December 2004

Estrogen plays an important role in many physiological events including carcinogenesis and the development of human breast cancer. However, the molecular mechanisms of estrogen signaling in cancers have not been clarified hitherto and accurate therapeutic prediction of breast cancer is earnestly desired. We first carried out estrogen-responsive expression profiling of approximately 9000 genes in estrogen receptor-positive human MCF-7 breast cancer cells. Based on the results, estrogen-responsive genes were selected for production of a custom-made cDNA microarray. Using a microarray consisting of the narrowed-down gene subset, we first analyzed the time course of the estrogen-responsive gene expression profiles in MCF-7 cells, resulting in subdivision of the genes up-regulated by estrogen into early-responsive and late-responsive genes. The expression patterns of several genes were confirmed by Northern blot analysis. We also analyzed the effects of the estrogen antagonists ICI 182780 and 4-hydroxytamoxifen (OHT) on the estrogen-responsive gene expression profiles in MCF-7 cells. While the regulation of most of the genes by estrogen was completely abolished by ICI 182780, some genes were partially regulated by estrogen even in the presence of OHT. Furthermore, the estrogen-responsive gene expression profiles of twelve cancer cell lines derived from the breast, ovary, stomach and other tissues were obtained and analyzed by hierarchical clustering including the profiles in MCF-7 cells. Several genes also showed up-regulation or down-regulation by estrogen in cell lines other than MCF-7 cells. The significance of the estrogen-responsive genes identified in these analyses concerning the nature of cancer is discussed.
Responsiveness of endometrial genes Connexin26, Connexin43, C3 and clusterin to primary estrogen, selective estrogen receptor modulators, phyto- and xenoestrogens
Authors: Heikaus S , Winterhager E , Traub O , Grummer R
Published: December 2004

Phytohormones and chemical compounds revealing estrogenic effects are of increasing interest for their possible influence on the physiology of the reproductive tract. The gap junction connexin (Cx) genes Cx26 and Cx43, the plasma glycoprotein clusterin gene and the complement C3 gene are highly regulated by estrogen in rat endometrium. To test the value of these genes as markers for estrogenic responsiveness we analyzed the effects of estradiol, diethylstilbestrol, the selective estrogen receptor modulators (SERMs) raloxifene and tamoxifen, the phytoestrogens genistein and daidzein, and the industrial compounds DDT (1,1,1-trichloro-2-(2-chlorophenyl)-2-(4-chlorophenyl) ethane) and polychlorinated biphenyl (PCB) on the transcription of these genes in rat endometrium in vivo. Enhancement of Cx26 and decrease of clusterin transcripts expression by estradiol was observed at 0.03 micro g/250 g body weight (BW), and induction of C3 expression was observed at 0.05 micro g/250 g BW. A comparable effect was obtained by a tenfold higher concentration of diethylstilbestrol. Tamoxifen had a regulatory effect on this set of genes at about a 300-fold higher concentration, while raloxifen revealed much weaker estrogenic activity. No effect on Cx43 transcripts was observed with any of the compounds at the concentrations used. An effect of genistein was observed only on Cx26 expression, while PCB decreased clusterin transcripts. These results show that Cx26, C3 and clusterin reveal a comparable sensitivity to estrogens and SERMs. With respect to the phytoestrogen genistein, however, Cx26 seems to be the most sensitive gene. The analysis of clusters of estrogen-sensitive endometrial genes could help to identify estrogenic substances, assess their potency, and elucidate their mechanism of action.
Role of Sp1 in insulin regulation of gene expression
Authors: Samson SL , Wong NC
Published: December 2004

Sp1 is a ubiquitous nuclear factor that plays a key role in maintaining basal transcription of 'house-keeping' genes. However, recent evidence points to a more important function for Sp1 in mediating 'cross-talk' between selected signaling cascades to regulate the target genes that respond to these pathways. The role of Sp1 in mediating the actions of the peptide hormone insulin is of specific interest and serves as a model for detailing effects of intracellular signaling on Sp1 activity. This review summarizes studies suggesting that changes in Sp1 phosphorylation provide one potential mechanism for manipulating activity of this protein. A growing body of evidence reveals that the DNA binding and transcription activity of Sp1 may increase or decrease in response to changes in phosphorylation. This enables 'fine-tuning' of Sp1 activity for regulation of gene transcription. Several mechanisms exist by which Sp1 alters gene activity in response to insulin. These include independent Sp1 activity as well as collaboration or competition with others factors. This review points to an ever-increasing role for Sp1 in regulating the transcription of genes in response to extracellular signals such as insulin.
Down but not out? A novel protein isoform of the estrogen receptor alpha is expressed in the estrogen receptor alpha knockout mouse
Authors: Kos M , Denger S , Reid G , Korach KS , Gannon F
Published: December 2004

The mouse knockout of the estrogen receptor alpha (ERalpha) gene, known as alphaERKO, has been extensively used for several years to study the role and function of ERalpha. Residual estradiol binding capacity in uterine tissue of 5-10% raised doubts if this knockout is a genuine null mutation of ERalpha. Although alternatively spliced ERalpha mRNA variants in the alphaERKO mouse were reported previously, the corresponding protein isoforms have not been detected to date. Here we show that a variant ERalpha protein, 61 kDa in size, is expressed in the uterine tissue of alphaERKO mice as a result of an alternative splicing. The transactivation capability of this protein is cell dependent and can be as high as 75% of the wild type ERalpha.
Oestrogenic regulation of an egr-1 transgene in rat anterior pituitary
Authors: Man PS , Carter DA
Published: December 2004

The C(2)H(2) zinc-finger transcription factor Egr-1 has previously been shown to play an essential role within the endocrine system as a molecular determinant of LH beta-subunit synthesis in anterior pituitary gonadotrophs. The extent to which Egr-1 may be a dynamic mediator of changes in gonadotroph function during the oestrous cycle is currently unclear. We have recently produced a novel rat transgenic model of egr-1 gene function in which proximal regions of the rat egr-1 gene drive expression of a reporter gene. In the present study, we have investigated the expression and physiological regulation of our egr-1/d4 enhanced green fluorescent protein (EGFP) transgene in the female rat pituitary gland. In situ hybridization analysis has revealed anterior pituitary-specific expression that is limited to a sub-population of cells that includes immunohistochemically defined gonadotrophs. Expression of the transgene is up-regulated 5-fold following ovariectomy. The transgene also exhibits regulated expression during the oestrous cycle, mRNA levels being significantly raised on pro-oestrus. Using an explant culture system, we have also demonstrated a direct stimulatory effect of 17beta-oestradiol on anterior pituitary transgene and egr-1 expression. The acute response of egr-1 to an oestrogenic stimulus is attenuated by the MEK (MAPK kinase) inhibitor U0126, and is accompanied by increased levels of phospho-p44/42 MAPK protein, suggesting regulation of egr-1 through a MAPK-linked pathway in the pituitary. These findings provide further evidence of cyclical endocrine regulation of egr-1 in the rat, demonstrate that proximal sequences of the egr-1 gene mediate endocrine-regulated expression, and indicate a novel pathway through which pituitary transcriptional responses to oestrogen may be mediated.
Inhibition of Src tyrosine kinase stimulates adrenal androgen production
Authors: Sirianni R , Carr BR , Ando S , Rainey WE
Published: December 2004

A unique characteristic of the primate adrenal is the ability to produce 19-carbon steroids, often called the adrenal androgens. Although it is clear that the major human adrenal androgens, dehydroepiandrosterone (DHEA) and DHEA sulfate (DHEA-S), are produced almost solely in the adrenal reticularis, the mechanisms regulating production are poorly understood. Herein, we tested the hypothesis that the Src family of tyrosine kinases are involved in the regulation of adrenal androgen production. The NCI-H295R human adrenal cell line and primary human adrenal cells in culture were used to study adrenal androgen production and expression of enzymes involved in steroidogenesis. To examine the role of Src tyrosine kinase, cells were treated with PP2, a specific Src inhibitor. Alternatively, adrenal cells were transfected with an expression vector containing a dominant-negative form of Src. PP2 treatment inhibited basal cortisol production while significantly increasing the production of DHEA and DHEA-S (together referred to as DHEA(S)) in both adrenal cell models. The effect of PP2 on steroidogenesis occurred along with a rapid induction of steroidogenic acute regulatory (StAR) protein synthesis as revealed by Western analysis. Treatment with PP2 also increased mRNA levels for StAR, and cholesterol side-chain cleavage (CYP11A) and 17alpha-hydroxylase/17,20-lyase (CYP17) enzymes. Treatment of adrenal cells with the cAMP agonist dibutyryladenosine cyclic monophosphate (dbcAMP), stimulated the production of cortisol and DHEA(S). However, treatment of adrenal cells with a combination of PP2 and dbcAMP enhanced the production of DHEA(S) while inhibiting cortisol production. During dbcAMP treatment PP2 was able to augment the expression of CYP17 and to inhibit the induction of 3beta-hydroxysteroid dehydrogenase type 2 (HSD3B2) levels. Increasing the CYP17 to HSD3B2 ratio is likely to promote the use of steroid precursors for the production of DHEA(S) and not for cortisol. Taken together these data suggest that the inhibition of Src tyrosine kinases causes adrenal cells to adopt a reticularis phenotype both by the production of DHEA(S) and by the steroidogenic enzymes expressed.
Tetracycline-regulated secretion of human insulin in a transfected non-endocrine cell line
Authors: Scougall KT , Maltin CA , Shaw JA
Published: December 2004

Long-term constitutive secretion of insulin by implantation of ex vivo transfected cells such as fibroblasts or myoblasts or in situ by intramuscular injection of naked plasmid DNA provides a potential approach to gene therapy for diabetes mellitus. A mechanism for regulating insulin secretion will be necessary to realize the therapeutic potential of this approach. A second obstacle is the inability of non-endocrine host cells to fully process proinsulin. Therefore, alteration of the wild-type cDNA will be necessary to achieve processing of proinsulin by endogenous endoproteases within these cells. The cDNAs for beta-galactosidase (beta), human wild-type proinsulin (hppI1) and a mutated construct (hppI4), in which the dibasic PC2 and PC3 cleavage sites had been altered to form furin cleavage sites, were sub-cloned into four vectors (pCR3, pVR1012, pIRES, pTRE), including a tetracycline responsive plasmid (pTRE) that requires co-transfection with another plasmid encoding a transactivator (pTet-off) for transgene expression. Transient transfection of the COS-7 fibroblast cell line with these constructs was performed using DEAE-dextran and liposomes. Analysis of vector efficiencies revealed that pTRE/pTet-off>pIRES>pCR3>pVR1012. Further analysis demonstrated total pro/insulin secretion of 2.33 ng/10(6) cells/24 h with > or =25% processed to insulin in hppI-1.pTRE/pTet-off-transfected cells compared with 0.39 ng/10(6) cells/24 h and >70% processing in hppI-4.pTRE/pTet-off-transfected cells. In co-transfection studies with pTRE-hppI1/pTet-off and pTRE-hppI4/pTet-off constructs, pro/insulin secretion was inhibited to 65-66% and 36-38% of control (100%) in the presence of 0.01 and 0.1 microg/ml tetracycline respectively over a 24-h incubation period. Furthermore, reversal of tetracycline inhibition was demonstrated for pTRE-hppI1/pTet-off- and pTRE-hppI4/pTet-off-transfected cells. After a 48-h incubation with 1.0 microg/ml tetracycline, total pro/insulin levels were 10 and 14% compared with untreated cells respectively. On tetracycline removal, total proinsulin levels increased and were equivalent to untreated groups 72 h later. In conclusion, regulation of fully processed human insulin secretion has been achieved in a transiently transfected non-endocrine cell line.
Adipose thyrotrophin receptor expression is elevated in Graves' and thyroid eye diseases ex vivo and indicates adipogenesis in progress in vivo
Authors: Starkey KJ , Janezic A , Jones G , Jordan N , Baker G , Ludgate M
Published: December 2004

The thyrotrophin receptor (TSHR) provides an autoantigenic link between the thyroid and orbit in Graves' (GD) and thyroid eye diseases (TED). We measured TSHR transcripts in different fat depots to determine whether TSHR expression levels are influenced by the autoimmune/inflammatory process and/or thyroid hormone status, using quantitative real-time PCR. Nine intact or fractionated adipose samples, from patients with GD and/or TED, were analysed ex vivo. Eight expressed the TSHR, at levels approaching the thyroid, and one was at the limit of detection. Thirteen/fifteen orbital and abdominal fat samples from patients free of GD and TED, measured ex vivo, were negative for TSHR transcripts and two were at the limit of detection. All preadipocyte samples induced to differentiate in vitro expressed the TSHR. To investigate the influence of thyroid hormone status on adipose TSHR expression, we induced hyper- and hypothyroidism in BALBc mice by administering tri-iodothyronine and propylthiouracil respectively. In euthyroid animals, whole fat samples were at the limit of detection and were not altered by thyroid hormone status. The results show that adipose TSHR expression ex vivo indicates adipogenesis in progress in vivo and is associated with the autoimmune/inflammatory process in GD and TED but is not restricted to the orbit or influenced by thyroid hormone status.
Transcriptional activation by estrogen receptor (ERalpha) and steroid receptor coactivator (SRC1) involves distinct mechanisms in yeast and mammalian cells
Authors: Sheppard HM , Matsuda S , Harries JC , Kindle KB , Heery DM
Published: December 2004

Steroid receptors activate transcription in yeast cells via interactions with endogenous coactivators and/or basal factors. We examined the effects of mutations in the ligand binding domain on the transcriptional activity of ERalpha in yeast. Our results show that mutations in Helix 3 (K366A) and Helix 12 (M547A, L548A) disrupt transcriptional activity of ERalpha in yeast, as previously observed in mammalian cells. However, replacement of a conserved tyrosine residue in Helix 12 with alanine or aspartate (Y541A and Y541D), which renders ERalpha constitutively active in mammalian cells, had only a weak stimulatory effect on ligand-independent reporter activation by ERalpha in yeast. Two-hybrid interaction experiments revealed that a Y541A mutant expressed in yeast was capable of ligand-independent binding to a mammalian coactivator, suggesting that there is a subtle difference in how this mutant interacts with mammalian and yeast cofactors. We also show that the ligand-dependent activities of ERalpha and progesterone receptor (PR) in yeast cells were strongly enhanced by the human p160 protein steroid receptor coactivator (SRC1), but not by CREB-Binding Protein (CBP) or the p300/CBP associated factor (P/CAF). Although the SRC1 activation domains AD1 and AD2 are functional in yeast, deletion of these sequences only partially impaired SRC1 coactivator function in this organism; this is in contrast to similar experiments in mammalian cells. Thus SRC1 sequences involved in recruitment of CBP/p300 and Co-Activator-Associated Arginine Methyltransferase (CARM-1) in mammalian cells are not essential for its function in yeast, suggesting that SRC1 operates via distinct mechanisms in yeast and mammalian cells.
Agonist-bound nuclear receptors: not just targets of coactivators
Authors: Fernandes I , White JH
Published: December 2004

Members of the nuclear receptor superfamily of ligand-regulated transcription factors are targets of a wide range of lipophilic signaling molecules as well as several drugs and xenobiotics that modulate many aspects of physiology and metabolism. Agonist binding to receptors is associated with recruitment of coactivators, which are essential for activation of target gene transcription. However, several biochemical and molecular genetic studies have shown that a full understanding of the function of agonist-bound receptors must also accommodate the recruitment of corepressors. These factors may attenuate agonist-induced transactivation, act more transiently as part of a cycle of cofactors recruited to target promoters by ligand-bound receptors, or function in hormone-dependent repression of target gene expression.
Functional interference between estrogen-related receptor alpha and peroxisome proliferator-activated receptor alpha/9-cis-retinoic acid receptor alpha heterodimer complex in the nuclear receptor response element-1 of the medium chain acyl-coenzyme A dehydrogenase gene
Authors: Maehara K , Hida T , Abe Y , Koga A , Ota K , Kutoh E
Published: December 2004

We undertook a study of molecular interference of nuclear orphan receptors. Nuclear receptor response element-1 (NRRE-1) from the human medium-chain acyl coenzyme A dehydrogenase (MCAD) gene promoter was shown to contain three hexamer elements (site 1 through 3) that are known to interact with a number of nuclear receptors including chicken ovalbumin upstream promoter transcription factor (COUP-TF) and estrogen-related receptor alpha (ERRalpha). We demonstrated that the peroxisome proliferator-activated receptor alpha/9-cis-retinoic acid receptor alpha (PPARalpha/RXRalpha) heterodimer complex can also bind to the two hexamer repeat sequences (between site 1 and site 3) arranged as an everted imperfect repeat separated by 14 bp (ER14). Mutations of the putative core elements have shown that these three sites are differentially involved in ERRalpha and PPARalpha/RXRalpha binding. Homodimer of ERRalpha was shown to interact between site 1 and site 3 (ER14). To date, no nuclear receptor is known to bind to response elements over such long intervals. Interestingly, site 1 was shown to be essential for ERRalpha binding while site 3 supports its binding only in the presence of site 1. Furthermore, it was shown that the binding profile of ERRalpha and PPARalpha/RXRalpha are competitive rather than making a high order complex within NRRE-1. At the cellular level, transcriptional activation driven by the PPARalpha/RXRalpha complex was counteracted by the expression of ERRalpha in HeLa cells. These results suggest that ERRalpha and PPARalpha/RXRalpha could interfere with each other's function through binding to similar DNA elements, thereby finetuning the transcriptional outcome of the target gene. Our findings suggest a mechanism whereby multiple nuclear receptors can activate or repress DNA binding or transcription via a single pleiotropic regulatory element.
Quantification of mRNA for the vitamin D metabolizing enzymes CYP27B1 and CYP24 and vitamin D receptor in kidney using real-time reverse transcriptase- polymerase chain reaction
Authors: Anderson PH , O'Loughlin PD , May BK , Morris HA
Published: December 2004

Critical to an understanding of the control of 1,25-dihydroxyvitamin D (1,25D) activity is a molecular appreciation of the regulation of three genes, 25-hydroxyvitamin D-1alpha-hydroxylase (CYP27B1), 25-hydroxyvitamin D-24-hydroxylase (CYP24) and vitamin D receptor (VDR). We now report the sensitivity, reproducibility and accuracy of a real-time reverse transcriptase-polymerase chain reaction protocol (Taqman) for the quantification of mRNA levels for these genes in total RNA extracted from kidney tIssue. The sensitivity of the protocol was at least 150 copies of mRNA per reaction. Reproducibility, expressed as the coefficient of variation, ranged between 14 and 30% at the level of approximately 10(4) copies of mRNA per reaction. Accuracy was estimated at greater than 95% for each of these mRNAs. This protocol allows for the comparison of absolute mRNA levels in extracted total RNA in kidneys from animals fed diets containing different levels of calcium, ranging from 0.05% to 1%. Serum 1,25D levels were decreased when the dietary calcium concentration was increased (P<0.05). The levels of CYP27B1 mRNA were highest in the animals fed the 0.05% calcium diet (P<0.01). Conversely, CYP24 and VDR mRNA levels were highest in the animals fed the 1% calcium diet (P<0.01). Both CYP27B1 and CYP24 mRNA levels were major determinants of serum 1,25D levels when dietary calcium intakes were varied in these adult animals (Multiple R(2)=0.70, P<0.01). No significant relationship was detected between kidney CYP27B1 and serum parathyroid hormone (PTH) suggesting that serum calcium may regulate CYP27B1 mRNA expression directly during normocalcaemia. Low levels of CYP24 mRNA were associated with high PTH levels. These findings suggest that kidney CYP24 activity, possibly regulated by factors such as PTH, acts in concert with kidney CYP27B1 to control serum 1,25D levels.
A single and novel natriuretic peptide is expressed in the heart and brain of the most primitive vertebrate, the hagfish (Eptatretus burgeri)
Authors: Kawakoshi A , Hyodo S , Yasuda A , Takei Y
Published: December 2004

In teleost fish and tetrapods, the natriuretic peptide (NP) family consists of ANP (atrial natriuretic peptide), BNP (brain natriuretic peptide) and VNP (ventricular natriuretic peptide) that are secreted from the heart, and C-type natriuretic peptide (CNP) that is found in the brain. However, CNP is the only NP identified in the heart and brain of elasmobranchs, suggesting that it is the ancestral type of the NP family and that ANP, BNP and VNP appeared later in the vertebrate phylogeny. To delineate more clearly the molecular evolution of this hormone family, we determined the sequence of NP molecule(s) in evolutionarily the oldest vertebrate group, the cyclostomes. We have cloned a novel NP cDNA from the heart and brain of hagfish, Eptatretus burgeri, using the RACE method and degenerate primers that amplify all known types of NP cDNAs. The novel NP, named EbuNP after the scientific name of this hagfish, appears to be the only NP in the heart and brain, as no other NP cDNAs were amplified even after specific removal of the cloned EbuNP mRNA from the mRNA pool, except for a minor alternatively spliced EbuNP cDNA with a truncated 3'-untranslated sequence. The EbuNP was equally similar to known NPs but was not considered to be a CNP because of the presence of a C-terminal tail sequence. The EbuNP gene was abundantly expressed in the cardiac atrium, ventricle, portal heart and brain but scarcely in the intestine; no expression was observed in the gill and kidney. Mass spectrometry of affinity-purified EbuNP in plasma, heart and brain revealed a 68 amino acid peptide circulating in the blood and stored in the heart, which is cleaved at the typical cleavage signal of a processing enzyme, furin, as observed in mammalian BNP. The C-terminal Gly residue was used for amidation as is the case in eel ANP. The immunoreactive EbuNP was not detected in the brain, suggesting the presence of a different processing form in the brain. These results show that the molecular evolution of the NP family in vertebrates is more complex than previously thought.
Interrenal steroid 21-hydroxylase in eels: primary structure, progesterone-specific activity and enhanced expression by ACTH
Authors: Li YY , Inoue K , Takei Y
Published: December 2004

Cytochrome P450 21-hydroxylase (P450c21) is a key enzyme for corticosteroidogenesis. To understand the regulatory mechanisms of cortisol production in fish, we have cloned a cDNA encoding P450c21, for the first time in non-mammalian vertebrates, from the head kidney of the eel (Anguilla japonica). The overall similarity of the deduced P450c21 sequence was modest (41-44% amino acid identity) between the eel and mammals. However, the functional domains for steroid-binding, heme-binding and proton-transfer sites were well conserved (74-100% identity). The eel P450c21 mRNA was expressed abundantly in the anterior quarter of the head kidney, but was undetectable in the remaining three-quarters or in other tissues including the gill, heart, liver, intestine, kidney, immature gonad and skeletal muscle. Functional expression of the cDNA clone in non-steroidogenic COS-1 cells produced a protein with high 21-hydroxylase activity to convert progesterone to 11-deoxycortisterone but not 17alpha-hydroxyprogesterone to 11-deoxycortisol, although the latter is a preferred substrate for mammalian P450c21. To examine whether 21-hydroxylated progesterone is actually 17alpha-hydroxylated in the eel interrenal, 11-deoxycorticosterone and (3)H-corticosterone were respectively incubated with the interrenal-containing anterior quarter of the head kidney. The separation of the steroids produced by two HPLC systems revealed that cortisol was produced from both substrates, showing the 17alpha-hydroxylation of 11-deoxycorticosterone and corticosterone in the eel interrenal. ACTH infused at 3 pmol/kg per min for 5 h consistently increased plasma cortisol levels and interrenal P450c21 mRNA levels in seawater eels. These results showed that the interrenal-specific eel P450c21 cloned in this study is involved in cortisol production through conversion of progesterone to 11-deoxycorticosterone in the interrenal-containing anterior quarter of eel head kidney. Furthermore, ACTH stimulates cortisol production in part through enhanced P450c21 expression in the eel interrenal.
Has the mechanism by which the endocrine placenta scavenges the mother whilst sparing the foetus been unmasked?
Authors: Lowry PJ
Published: December 2004

The endocrine placenta has a dilemma; it shares the foetal circulation and yet it needs to secrete active peptide hormones into the maternal circulation to control her metabolism to meet the demands of the growing foetus. Simultaneously, it needs to allow the endocrine systems of the foetus to develop independently. This Article will describe how peptide hormones are processed from inactive intermediates and will propose a hypothesis of how the placenta has revised this process to protect the foetus from the potentially damaging affects of these products.
The effects of GH-releasing hormone/somatostatin on the 5'-promoter activity of the GH gene in vitro
Authors: Morishita M , Iwasaki Y , Onishi A , Asai M , Mutsuga N , Yoshida M , Oiso Y , Inoue K , Murohara T
Published: December 2004

The two hypothalamic hormones, GH-releasing hormone (GHRH) and somatostatin (SRIF), are known to regulate GH secretion. However, the effects of these hormones on GH gene expression are not completely clear, partly because of the lack of appropriate host cells maintaining the original characteristics of the somatotroph. Since MtT/S, a pure somatotroph cell line, has become available, the effects of GHRH and SRIF on GH gene transcription have been studied using a subclone of MtT/S (MtT/SGL), in which the GH gene 5'-promoter-luciferase fusion gene was stably incorporated. The expression of GHRH receptor and SRIF receptor subtypes was also studied by RT-PCR. The results showed that MtT/SGL cells intrinsically expressed the functional GHRH receptor and all of the SRIF receptor subtypes. The expression of GHRH receptor was markedly enhanced by glucocorticoid pretreatment and, in the presence of corticosterone and 3-isobutyl-1-methylxanthine, GHRH (at or above 100 pM) stimulated GH gene 5'-promoter activity in a dose-dependent manner. On the other hand, SRIF (100 nM) significantly antagonized the effect of GHRH, which was completely reversed by pretreatment with pertussis toxin (50 ng/ml). Taken together, the present data indicated that both GHRH and SRIF are involved in the transcriptional regulation of the GH gene, and that the effect of SRIF is mediated through pertussis toxin-sensitive G protein. The MtT/SGL cell line is a good in vitro model for studying the molecular mechanisms of GH gene transcription by GHRH and/or SRIF.
Threshold effects of glucose transporter-4 (GLUT4) deficiency on cardiac glucose uptake and development of hypertrophy
Authors: Kaczmarczyk SJ , Andrikopoulos S , Favaloro J , Domenighetti AA , Dunn A , Ernst M , Grail D , Fodero-Tavoletti M , Huggins CE , Delbridge LM , Zajac JD , Proietto J
Published: December 2004

The aim of this study was to investigate the metabolic and structural consequences of a decrease in glucose transporter-4 (GLUT4) levels on the heart. The CreLoxP system was utilised to delete GLUT4 in muscle tIssue including heart. The presence of the PGK-neoR cassette in the GLUT4-Lox mice resulted in reduced expression in all tIssues to levels 15-30% of wild-type control mice. In mice expressing Cre recombinase, there was a further reduction of GLUT4 in cardiac tIssue to almost undetectable levels. Cardiac glucose uptake was measured basally and during a euglycaemic/hyperinsulinaemic clamp using 2-deoxy-[1-(14)C]glucose. Insulin-stimulated glucose uptake was normal in hearts expressing 15% of normal GLUT4 levels but markedly reduced in mice with more profound reduction in GLUT4. Cardiac enlargement occurred only when GLUT4 levels were less than 5% of normal values. In heart there is a threshold level of GLUT4 above which insulin-stimulated glucose uptake is maintained. As little as 5% of normal GLUT4 levels expressed in heart is sufficient to prevent the development of cardiac hypertrophy.
Identification of dimeric and oligomeric complexes of the human oxytocin receptor by co-immunoprecipitation and bioluminescence resonance energy transfer
Authors: Devost D , Zingg HH
Published: December 2004

The nonapeptide hormone oxytocin exerts many important biological functions, including uterine contractions during parturition and milk ejection during lactation. The manifold effects of oxytocin are mediated by a single oxytocin receptor (OTR) type, a member of the super-family of G-protein-coupled receptors. There is accumulating recent evidence that certain G-protein-coupled receptors exist in the form of oligomeric complexes. Here we demonstrate, using two different co-immunoprecipitation strategies as well as bioluminescence resonance energy transfer techniques, that the OTR is capable of forming oligomeric complexes in vivo and that these complexes exist at the cell surface membrane. The human OTR was N-terminally tagged with either a Myc or Flag epitope and transiently expressed in COS-7 cells. Cell lysates were immunoprecipitated using an anti-Flag antibody and analyzed by SDS-PAGE and Western blotting using an anti-Myc antibody, or vice versa. Either strategy provided evidence for the co-precipitation of Myc- or Flag-tagged OTR respectively.Biochemical characterization of OTR dimers showed that homodimer formation is not dependent on the establishment of disulfide bonds. The existence of OTR dimers and oligomers at the level of the cell surface was demonstrated by exposing intact living cells to an anti-Flag antibody and analyzing the immunoprecipitate by Western blotting with an anti-Myc antibody. This approach demonstrated furthermore that the presence of receptor oligomers at the cell surface is modulated by ligand in a time-dependent fashion. Finally, we obtained evidence that the OTR is forming oligomeric structures in intact living cells by observing the occurrence of bioluminescence resonance energy transfer in cells co-transfected with OTR constructs bearing at their C-terminus either a Renilla luciferase or the yellow fluorescent protein. Taken together, these data show that the OTR can form homodimers and oligomers in the cell model used and that these oligomers are present at the cell surface.
Regulation of transcripts encoding ADAMTS-1 (a disintegrin and metalloproteinase with thrombospondin-like motifs-1) and progesterone receptor by human chorionic gonadotropin in equine preovulatory follicles
Authors: Boerboom D , Russell DL , Richards JS , Sirois J
Published: December 2004

One member of a new family of metalloproteinases, a disintegrin and metalloproteinase with thrombospondin-like motifs-1 (ADAMTS-1), has been found to be expressed and hormonally induced in granulosa cells of ovulating rodent follicles. Furthermore, the targeted disruption of the ADAMTS-1 gene resulted in ovarian defects associated with severely impaired fertility. While these data demonstrate the importance of ADAMTS-1 in rodent ovarian physiology, the potential role of ADAMTS-1 in the ovulatory process of monoovulatory species remains unknown. The objectives of this study were to clone the equine ADAMTS-1 primary transcript and to study its regulation during human chorionic gonadotropin (hCG)-induced ovulation. A 3573 bp follicular cDNA library clone was isolated and found to encode a nearly complete, highly conserved ADAMTS-1 homologue. Real-time RT-PCR analysis detected this transcript in diverse tIssues, including previously unreported sites of ADAMTS-1 expression such as the male reproductive tract, the follicular theca interna and the mature corpus luteum. The tIssue distribution of the progesterone receptor (PR), a known regulator of ADAMTS-1 expression in rodent preovulatory follicles, was found to overlap that of ADAMTS-1 in some tIssues. A study of the regulation of follicular ADAMTS-1 and PR mRNAs during the hCG-induced ovulatory process revealed distinct patterns of regulation in granulosa cells and in theca interna. In granulosa cells, ADAMTS-1 mRNA was found to be induced at 12 h post-hCG (P<0.05), followed by a return to basal levels by 30 h and a re-increase at 33-39 h (P<0.05). A concomitant increase in PR mRNA (P<0.05) was observed at 12 h post-hCG. In theca interna, abundant ADAMTS-1 mRNA was detected at all timepoints, and levels increased transiently at 33 h post-hCG (P<0.05), whereas no significant change was observed in PR mRNA. Together, these data demonstrate for the first time the hormonally regulated ovarian expression of ADAMTS-1 in a monoovulatory species, and identify a novel biphasic regulation of ADAMTS-1 in granulosa cells and a regulated expression in theca interna that were not previously observed in rodents.
The recovery of some components of the renin angiotensin system in the rat pancreas after chronic exposure to hypoxic condition
Authors: Ip SP , Wong TP , Tsai SJ , Leung PS
Published: December 2004

Previous studies have shown that the expression of the major components from a local pancreatic renin-angiotensin system (RAS) was upregulated after chronic exposure to oxygen deprivation (10% oxygen). In the present study, the reversibility of expression for the pancreatic RAS affected by chronic hypoxia was investigated in the pancreas. Rats were first subject to hypoxia for one Month and they were then returned to normoxic conditions for a varying period of time (1, 2, 3 and 4 weeks). The degree of recovery in the expression of RAS components was analyzed with standard curve-quantitative competitive-reverse transcription-polymerase chain reaction (SC-QC-RT-PCR), Western blot analysis and a specific assay for angiotensin-converting enzyme (ACE) activity. Results from SC-QC-RT-PCR showed that the upregulated expression of angiotensin II type 1 (AT(1)) receptor mRNA following chronic hypoxia could be completely restored to the control level after the rats were returned to the normoxic condition for 3 weeks. The reversibility of mRNA expression for angiotensin II type 2 (AT(2)) receptor and angiotensinogen was observed after the return to normoxic conditions for 2 and 3 weeks respectively when compared with that of their respective controls. Results from Western blot analysis further confirmed that the expression of AT(1) receptor protein was also reversible after return to normoxic conditions for 4 weeks. In addition, the activation of ACE activity returned to its normal level in a time-dependent manner. These data indicate that the upregulation of a local pancreatic RAS affected by chronic hypoxia could be recoverable. The significance of its reversibility and adaptability following chronic hypoxia may be of physiological relevance to the pancreas.
Identification and characterization of a cDNA and the gene encoding the mouse ubiquitously expressed glucose-6-phosphatase catalytic subunit-related protein
Authors: Boustead JN , Martin CC , Oeser JK , Svitek CA , Hunter SI , Hutton JC , O'Brien RM
Published: December 2004

Glucose-6-phosphatase (G6Pase) catalyzes the final step in the gluconeogenic and glycogenolytic pathways, the hydrolysis of glucose-6-phosphate (G6P) to glucose and phosphate. This paper describes the identification and characterization of a cDNA and the gene encoding the mouse ubiquitously expressed G6Pase catalytic subunit-related protein (UGRP). The open reading frame of this UGRP cDNA encodes a protein (346 amino acids (aa); Mr 38,755) that shares 36% overall identity (56% similarity) with the mouse G6Pase catalytic subunit (357 aa; Mr 40,454). UGRP exhibits a similar predicted transmembrane topology and conservation of many of the catalytically important residues with the G6Pase catalytic subunit; however, unlike the G6Pase catalytic subunit, UGRP does not catalyze G6P hydrolysis and does not contain a carboxy-terminal di-lysine endoplasmic reticulum retention signal. UGRP mRNA was detected by RNA blot analysis in every mouse tissue examined with the highest expression in heart, brain, testis and kidney. Database analysis showed that the mouse UGRP gene is composed of six exons, spans approximately 4.2 kbp of genomic DNA and is located on chromosome 11 along with the G6Pase catalytic subunit gene. The UGRP gene transcription start sites were mapped by primer extension analysis, and the activity of the mouse UGRP gene promoter was analyzed using luciferase fusion gene constructs. In contrast to the G6Pase catalytic subunit gene promoter, the UGRP promoter was highly active in all cell lines examined.
Postprandial lipemia is modified by the presence of the polymorphism present in the exon 1 variant at the SR-BI gene locus
Authors: Perez-Martinez P , Lopez-Miranda J , Ordovas JM , Bellido C , Marin C , Gomez P , Paniagua JA , Moreno JA , Fuentes F , Perez-Jimenez F
Published: December 2004

It has recently been reported that carriers of the less common allele at the scavenger receptor class B type I (SR-BI) exon 1 polymorphism are more susceptible to the presence of saturated fatty acid in the diet because of a greater increase in LDL cholesterol. Our aim was to determine if this polymorphism could also influence postprandial lipoprotein metabolism, because the SR-BI has been described as a possible mediator in the intestinal absorption of triacylglycerols. Forty-seven normolipidemic volunteers who were homozygous for the E3 allele at the APOE gene were selected [37 homozygous for the common genotype (1/1) at the SR-BI exon 1 polymorphism and 10 heterozygous (1/2)]. They were given a fat-rich meal containing 1 g fat and 7 mg cholesterol per kg body weight and vitamin A 60,000 IU/m2 body surface. Fat accounted for 60% of calories, and protein and carbohydrates accounted for 15% and 25% of energy respectively. Blood samples were taken at time 0, every 1 h until 6 h, and every 2.5 h until 11 h. Total cholesterol and triacylglycerols in plasma, and cholesterol, triacylglycerols and retinyl palmitate in triacylglycerol-rich lipoproteins (large and small triacylglycerol-rich lipoproteins) were determined. Postprandial responses for triacylglycerols and retinyl palmitate in small triacylglycerol-rich lipoproteins were higher in 1/1 individuals than in 1/2 individuals. No other significant differences were noted. Our data show that the presence of the genotype 1/2 is associated with a lower postprandial lipemic response.
Estrogen receptor-alpha interaction with the CREB binding protein coactivator is regulated by the cellular environment
Authors: Jaber BM , Mukopadhyay R , Smith CL
Published: December 2004

The p160 coactivators, steroid receptor coactivator-1 (SRC-1), transcriptional intermediary factor-2 (TIF2) and receptor-associated coactivator-3 (RAC3), as well as the coactivator/integrator CBP, mediate estrogen receptor-alpha (ERalpha)-dependent gene expression. Although these coactivators are widely expressed, ERalpha transcriptional activity is cell-type dependent. In this study, we investigated ERalpha interaction with p160 coactivators and CBP in HeLa and HepG2 cell lines. Basal and estradiol (E2)-dependent interactions between the ERalpha ligand-binding domain (LBD) and SRC-1, TIF2 or RAC3 were observed in HeLa and HepG2 cells. The extents of hormone-dependent interactions were similar and interactions between each of the p160 coactivators and the ERalpha LBD were not enhanced by 4-hydroxytamoxifen in either cell type. In contrast to the situation for p160 coactivators, E2-dependent interaction of the ERalpha LBD with CBP or p300 was detected in HeLa but not HepG2 cells by mammalian two-hybrid and coimmunoprecipitation assays, indicating that the cellular environment modulates ERalpha-CBP/p300 interaction. Furthermore, interactions between CBP and p160 coactivators are much more robust in HeLa than HepG2 cells suggesting that poor CBP-p160 interactions are insufficient to support ERalpha-CBP-p160 ternary complexes important for nuclear receptor-CBP interactions. Alterations in p160 coactivators or CBP expression between these two cell types did not account for differences in ERalpha-p160-CBP interactions. Taken together, these data revealed the influence of cellular environment on ERalpha-CBP/p300 interactions, as well as CBP-p160 coactivator binding, and suggest that these differences may contribute to the cell specificity of ERalpha-dependent gene expression.