bluuebunny/crossref_metadata_2025 huggingface dataset:
Regulation of thyrotropin receptor protein expression in insect cells
Authors: Seetharamaiah GS , Kaithamana S , Desai RK , Prabhakar BS
Published: December 2004

Expression of large quantities of conformationally intact thyrotropin receptor (TSHR) is essential to understand the structure-function relationship of the receptor. We expressed three different constructs of full-length human TSHR in insect cells: (a) a TSHR cDNA lacking signal sequence (TSHR-ns), (b) a TSHR cDNA containing human TSHR signal sequence (TSHR-hs) and (c) a TSHR cDNA with baculovirus envelope protein encoded signal sequence gp-67 (TSHR-gp). No unique protein band, corresponding to any of these recombinant proteins, was visible upon Coomassie Blue staining after SDS-PAGE. However, Western blot using TSHR specific monoclonal antibody showed unique bands around 80, 100 and 100 kDa in TSHR-ns, TSHR-hs and TSHR-gp virus infected insect cells respectively. All three full-length TSHR proteins could neutralize the TSH binding inhibitory immunoglobulin (TBII) activity from sera of experimental animals. However, only glycosylated proteins (TSHR-hs and TSHR-gp) neutralized the TBII activity of sera from autoimmune thyroid patients, confirming the importance of glycosylation for patient autoantibody reactivity. Expression levels of full-length TSHR proteins were much lower than the levels of similarly produced corresponding ectodomains of TSHR proteins. Southern blot and Northern blot analyses showed that DNA and RNA levels in full-length TSHR virus infected insect cells were comparable to the levels found in cells infected with viruses encoding only the ectodomain of TSHR. These data suggest that full-length TSHR expression is very low and is regulated at the translational level.
Variant estrogen receptor-alpha messenger RNA expression in hormone-independent human breast cancer cells
Authors: Coutts AS , Leygue E , Murphy LC
Published: December 2004

T5-PRF cells are insensitive to the growth-stimulatory effects of estrogen while still retaining expression of estrogen receptor-alpha (ER-alpha). In the apparent absence of ligand, T5-PRF cells have a 3. 6+/-0.5 (s.e.m.)-fold increased basal ER-alpha activity and elevated basal progesterone receptor levels compared with the parent, estrogen-sensitive, T5 cells. Long-range ER-alpha reverse transcription-PCR was performed to characterize variant ER-alpha mRNA expression in the two cell lines. An increased relative expression of an exon 3/4-deleted ER-alpha mRNA variant was found in T5-PRF. Recombinant expression of this ER-alpha variant resulted in significantly increased estrogen responsiveness, as well as a trend to increased basal ligand-independent activity when expressed with wild-type ER-alpha in ER-negative cell lines, as well as significantly increasing both ligand-independent and estrogen-induced ER-alpha transcriptional activity when expressed in parental T5 cells. These results suggest a role for altered variant ER-alpha in ligand-independent activation of ER-alpha which may contribute to hormone independence in breast tumors.
Analysis of binding properties between 20 kDa human growth hormone (hGH) and hGH receptor (hGHR): the binding affinity for hGHR extracellular domain and mode of receptor dimerization
Authors: Uchida H , Banba S , Wada M , Matsumoto K , Ikeda M , Naito N , Tanaka E , Honjo M
Published: December 2004

It has recently been shown that 20 kDa human growth hormone (hGH) forms the 1:2 hGH:hGH receptor (hGHR) complex and expresses full agonistic activity, although it hardly forms the 1:1 GH:GHR complex as compared with 22 kDa hGH. To clarify this mechanism, we analyzed the mode of receptor dimerization of 20 kDa hGH using the intact form and mutants. Complex formation analysis between hGHR extracellular domain (hGHBP) and either site1 mutant (K157A) or site2 mutant (G105R) by gel-filtration showed that the site1 mutant apparently formed no 1:1 complex and that the site2 mutant formed only the 1:1 complex. Cell proliferation analysis revealed that the activity curve (vs ligand concentration) of 20 kDa hGH showed a bell-shaped pattern. This indicates that the receptor dimerization of 20 kDa hGH proceeds in a sequential manner. Based on this sequential binding we have produced a mathematical model for receptor dimerization as a function of [hGH], [hGHBP], K(d) values for the first hGHBP binding (K(d1)) and the second hGHBP binding (K(d2)). The result of 20 kDa hGH binding to (S201C) hGHBP immobilized on biosensor tip showed that the K(d1) value was 1. 6x10(-8) M. Adopting this value as a constant in the function described above, we have obtained calculative hGHR dimerization curves vs hGH concentration. Since the K(d2) value could not be experimentally determined, the curves were simulatively obtained with varied K(d2) values. The simulated curve pattern coincided with the experimental result of the cell proliferation in Ba/F3-hGHR when the value 2.5x10(-10) M was adopted as K(d2). In conclusion, although the affinity of 20 kDa hGH for the first hGHR binding is reduced to one-tenth, that for the second binding is increased ten-fold in comparison with those of 22 kDa hGH, indicating that 20 kDa hGH can be an effective hGH isoform in the presence of hGHBP.
Analysis of the 5'-upstream regions of the human relaxin H1 and H2 genes and their chromosomal localization on chromosome 9p24.1 by radiation hybrid and breakpoint mapping
Authors: Garibay-Tupas JL , Csiszar K , Fox M , Povey S , Bryant-Greenwood GD
Published: December 2004

Relaxins are known endocrine and autocrine/paracrine hormones that play a major role in reproduction. In the human there are two relaxin genes, H1 and H2 which share 90% sequence homology within their coding region. The biological and evolutionary significance of two highly homologous and biologically active human relaxins is unknown. In order to achieve a better understanding of the regulatory mechanisms involved in the differential expression of these two genes and to gain insight into their role(s) in the preterm premature rupture of the membranes, we have investigated the properties of their 5'-upstream regions and mapped them both by radiation hybrid and breakpoint mapping into the same chromosome 9p24.1 locus. The 5' ends of these relaxin genes could be divided into a proximal highly homologous segment and a distal non-homologous region. Within the proximal region are contained several putative regulatory elements common to both genes, suggesting a similar regulatory mechanism. The clustering of the relaxin genes within the same chromosomal locus suggests that these genes may be under a common regulation. On the other hand, a distinct gene-specific regulation may also exist for the individual relaxin genes since cis elements specific to each gene were identified at their 5' ends. Moreover, the observed divergence at the distal region of their 5'-upstream sequences may provide the structural features that act as gene-specific transcription regulators. Since the two genes are highly homologous in both their coding and flanking regions, the divergence at the distal region of their 5' ends may be important in the regulation of these genes and in their involvement in the pathology of preterm birth.
Subtilase-like pro-protein convertases: from molecular specificity to therapeutic applications
Authors: Bergeron F , Leduc R , Day R
Published: December 2004

Limited proteolysis of most large protein precursors is carried out in vivo by the subtilisin-like pro-protein convertases. Many important biological processes such as peptide hormone synthesis, viral protein processing and receptor maturation involve proteolytic processing by these enzymes, making them potential targets for the development of novel therapeutic agents. However, the efficient development of such molecules requires a better understanding of the molecular mechanisms of proteolytic protein processing. Herein, we review the most recent findings on the molecular aspects of subtilisin-like convertase activity, such as the structural analysis of the proteases, the mechanisms of enzyme/substrate specificity, their interaction with other proteins such as 7B2, and the comparative tissue and cellular distribution of the enzymes and their substrates. These data are then used as a background for the review of the known biological functions of subtilisin-like pro-protein convertases, the reported clinical cases involving proteolytic processing defects and, finally, the ongoing development of new therapeutic inhibitor molecules based on this knowledge.
Urocortin is the principal ligand for the corticotrophin-releasing factor binding protein in the ovine brain with no evidence for a sauvagine-like peptide
Authors: Baigent SM , Lowry PJ
Published: December 2004

To purify novel ligands for the corticotrophin-releasing factor binding protein (CRF-BP) from ovine brain, whole brain was homogenised in methanol and the supernatant extracted on Sep-pak C18 cartridges followed by a preliminary HPLC step. Three peaks of ovine CRF-BP ligand activity were detected in the HPLC fractions, the first two of which were also detected by a specific corticotrophin-releasing factor two-site immunoradiometric assay, the third peak being detected by a human CRF-BP ligand assay, which will not detect ovine CRF. Human CRF-BP ligand-containing fractions were further purified by affinity chromatography on a human recombinant CRF-BP column with two additional HPLC steps. The human CRF-BP ligand was found to: (a) possess a molecular mass of 4707 Daltons, (b) have an N-terminal amino acid sequence (5 residues) identical to rat urocortin, (c) be detected by a specific urocortin radioimmunoassay, (d) have high affinity for both the human and ovine CRF-BPs and (e) be present in many regions of the ovine brain. Additionally, a 300 bp cDNA fragment sharing 83% homology with the rat urocortin gene was cloned from ovine brain, the product of which was predicted to have an identical amino acid sequence to that of rat urocortin. These pieces of information confirmed the identity of the human CRF-BP ligand as an ovine urocortin. The specially developed CRF-BP ligand assays showed that the rank orders of affinity of the CRF family members for human CRF-BP were: carp urotensin-1>>human CRF=rat/ovine urocortin>human urocortin>>frog sauvagine>>ovine CRF, and those for the ovine CRF-BP were: carp urotensin-1> human CRF=rat/ovine urocortin>human urocortin> frog sauvagine>>ovine CRF. This study describes a successful technique for the purification and detection of peptide ligands for the CRF-BP. We conclude that urocortin is the principal ligand for the CRF-BP in ovine brain and we could find no evidence for a centrally located mammalian sauvagine-like peptide.
Phenotypic variability and origins of mutations in the gene encoding 3beta-hydroxysteroid dehydrogenase type II
Authors: McCartin S , Russell AJ , Fisher RA , Wallace AM , Arnhold IJ , Mason JI , Varley J , Mendonca BB , Sutcliffe RG
Published: December 2004

Mutations in HSD3B2, the gene for 3beta-hydroxysteroid dehydrogenase type II (3beta-HSD II) have been detected and activities analysed through the in vitro expression of mutant cDNAs. Two full sibs with male pseudohermaphroditism were found to be double heterozygotes: N100S/266DeltaA. This genotype leads to the most profound loss of 3beta-HSD II enzyme activity (1.3% of normal) described to date in cases without severe salt-loss. One sib (N100S/266DeltaA) is the first reported male case of type II deficiency affected with premature adrenarche. Three apparently independent kindreds had propositi affected with the HSD3B2 mutation A82T/A82T, which is associated with a non salt-losing phenotype with variable expressivity in females. These three families had the same extended HSD3B haplotype and are likely to have inherited the same ancestral mutation. The significance of this finding is discussed in the light of the presence of A82T mutation at a homologous position in pseudogene varphi5 that is present in the HSD3B cluster.
Sodium butyrate/retinoic acid costimulation induces apoptosis-independent growth arrest and cell differentiation in normal and ras-transformed seminal vesicle epithelial cells unresponsive to retinoic acid
Authors: Buommino E , Pasquali D , Sinisi AA , Bellastella A , Morelli F , Metafora S
Published: December 2004

Retinoic acid (RA) and sodium butyrate (NaB) are regulators of cell growth and differentiation. We studied their effect on normal (SVC1) or v-Ki-ras-transformed (Ki-SVC1) rat seminal vesicle (SV) epithelial cell lines. The treatment of these cells with 10(-((7( M RA did not produce significant changes in the morphological and biochemical parameters analyzed. When RA was used in combination with 2 mM NaB, the treatment induced substantial morphological changes, apoptosis-independent growth arrest, up-regulation of tissue transglutaminase (tTGase), and down-regulation of beta and gamma RA receptor (RAR) mRNA expression. The same cells did not express RAR alpha either before or after NaB/RA treatment. A similar treatment did not change the amount of mRNA coding for the protein SV-IV (a typical differentiation marker of the SV epithelium) in normal or ras-transformed cells nor the level of v-Ki-ras mRNA in Ki-SVC1 cells. These findings suggest that a defective RA/RARs signaling pathway is probably the biochemical condition that underlies the unresponsiveness to RA of our in vitro culture system, and indirectly points to the possibility that the NaB/RA-induced effects were brought about by a cooperation at the transcription level between the histone deacetylase inhibitory activity of NaB and the ability of RA/RAR to modulate the expression of various genes involved in the control of cell growth and differentiation.
Acute regulation of the bovine gene for the steroidogenic acute regulatory protein in ovarian theca and adrenocortical cells
Authors: Ivell R , Tillmann G , Wang H , Nicol M , Stewart PM , Bartlick B , Walther N , Mason JI , Morley SD
Published: December 2004

Upregulation of the steroidogenic acute regulatory protein (StAR) is implicated in the rapid synthesis and secretion of steroidogenic cells to produce steroids in response to stimulation by trophic hormones of the gonadal and stress axes. In the present study, we have assessed the kinetics of both StAR gene transcription and protein biosynthesis in primary cell cultures of bovine adrenocortical and ovarian theca cells, under conditions of acute stimulation by corticotrophin (ACTH) and luteinizing hormone (LH), respectively. In both cell systems, detectable upregulation of StAR gene transcription occurred within 1-2 h, reaching maxima at 4 h (theca cells) or 6 h (adrenocortical cells). mRNA levels returned rapidly to baseline, by 12 h or 24 h, respectively. Specific StAR protein levels were assessed by western blotting using a novel antibody raised against a bovine StAR peptide, and showed a similar fast upregulation, albeit delayed by 1-2 h compared with the mRNA. The response of the cultured theca cells was more acute than that of the adrenocortical cells, possibly reflecting the propensity of the LH receptor to desensitize rapidly, unlike the ACTH receptor. The primary bovine theca cell cultures were also used for fully homologous transfection studies using various deletion promoter-reporter constructs of the bovine StAR gene. Kinetic analysis of the results indicated that the acute transcriptional response resides within the proximal (-315 bp) promoter region, which includes two putative responsive elements for the steroidogenic factor-1. More distal promoter regions may be involved in modulating the specificity of expression by combining enhancer and inhibitory functions.
Thyroid hormone deiodination in the domestic cat
Authors: Foster DJ , Thoday KL , Beckett GJ
Published: December 2004

We have investigated thyroid hormone deiodination in the liver, kidney and thyroid of the domestic cat. Affinity labelling with (125)I-bromoacetyl reverse T(3) (125)(I-BrAc-rT(3) demonstrated that liver and kidney, but not the thyroid, express type I iodothyronine deiodinase (IDI), results that were confirmed by measuring the activity of the IDI using (125)I-rT(3) and T(4) as substrate. Feline hepatic and renal IDI metabolised rT(3) at approximately 0.2% of the rate of rat hepatic IDI under identical assay conditions. The K(m) of the feline enzyme was at least 500-fold greater than that of rat IDI. However, feline and rat hepatic IDI metabolised T(4) at a similar rate and had similar K(m) values (1.35 microM and 2.25 microM, respectively). This study demonstrates that cats and rats express IDI in the liver and kidney in similar concentrations; however, the feline enzyme appears unable to utilise rT(3) as a substrate under physiological conditions.
Differential expression of 17beta-hydroxysteroid dehydrogenases types 2 and 4 in human endometrial epithelial cell lines
Authors: Husen B , Psonka N , Jacob-Meisel M , Keil C , Rune GM
Published: December 2004

In the endometrium two enzymes are known to convert estradiol to its inactive metabolite estrone: microsomal 17beta-hydroxysteroid dehydrogenase type 2 (17beta-HSD2) and peroxisomal 17beta-HSD4. In order to elucidate the particular function of each of these two different enzymes, the human endometrial epithelial cell lines HEC-1-A and RL95-2 were examined with respect to the expression of 17betaHSD isozymes. They were compared with human endometrium in vivo. Non-radioactive in situ hybridization revealed both enzymes in glandular epithelial cells of human endometrium. The two cell lines were screened for mRNA expression of 17beta-HSD 1-4 by RT-PCR and Northern blot. 17beta-HSD2 and 4 could be detected by either method, 17beta-HSD1 only by RT-PCR, 17beta-HSD3 not at all. Both cell lines were proven to have no receptor for progesterone which is known as a physiological inducer of several 17beta-HSD isozymes. To study the regulation of 17beta-HSD2 and 17betaHSD4, the concentration of fetal calf serum in the cell culture media was reduced stepwise to 0.3% by dilution with a defined serum replacement. This treatment led to an inhibition of 17beta-HSD2 mRNA expression and an increase in the mRNA expression of 17beta-HSD4. Concomitantly, distinct morphological changes were observed, such as a decrease in the number and length of microvilli and a decrease in the formation of domes on top of the monolayers. The endometrial epithelial cell lines HEC-1-A and RL95-2 represent a suitable in vitro model for further studies of the differential expression of the major endometrial HSD isozymes, independent of the effect of progesterone.
Biological activity of single chain chorionic gonadotropin, hCGalphabeta, is decreased upon deletion of five carboxyl terminal amino acids of the alpha subunit without affecting its receptor binding
Authors: Sen Gupta C , Dighe RR
Published: December 2004

The strategy of translationally fusing the two subunits of human chorionic gonadotropin (hCG) has been used to produce recombinant single chain hCG in which the C-terminus of the alpha subunit is fused to the N-terminus beta without any linker using Pichia pastoris expression system. The Pichia-expressed hCGalphabeta (phCGalphabeta) attained an overall conformation similar to that of hCG, and could bind to the receptor and elicit biological response, suggesting that receptor binding and signal transduction can take place even with a molecule having blocked the C-terminus of the alpha subunit. The carboxyl terminal of the alpha subunit has been shown to be involved in hormone binding and signal transduction of all the heterodimeric glycoprotein hormones. However, deletion of five amino acids from the C-terminus of the alpha subunit in the single chain hCG did not alter the overall conformation of the fusion molecule and its receptor binding ability, but led to a significant reduction in its ability to elicit biological response. These data show that these five amino acids at the C-terminus of the alpha subunit in the single chain hCG are not absolutely essential for attaining a conformation required for receptor binding, but are essential for obtaining a full biological response.
Expression of the prolactin receptor (tiPRL-R) gene in tilapia Oreochromis niloticus: tissue distribution and cellular localization in osmoregulatory organs
Authors: Sandra O , Le Rouzic P , Cauty C , Edery M , Prunet P
Published: December 2004

The expression of the prolactin receptor (PRL-R) gene has been investigated in various tissues of tilapia (Oreochromis niloticus) reared in fresh or brackish water. Using a cDNA probe spanning the extracellular domain of the tilapia PRL-R and Northern blot analysis, the presence of tilapia PRL-R mRNA has been confirmed in the osmoregulatory organs and has been detected in other tissues, including the skin, the brain, the reproductive organs, and the two major hematopoietic organs (spleen and head kidney), as well as circulating lymphocytes. These findings suggest a conservation of the physiological processes regulated by prolactin throughout the vertebrates, including immunity and central nervous activity. A non-radioactive in situ hybridization procedure has allowed us to detect the expression of the tilapia PRL-R in the branchial chloride cells and the intestinal mucosal layer of fresh water animals, confirming the direct control exerted by prolactin on the water and ionic exchanges in tilapia. In all the tissues examined one unique PRL-R transcript has been detected with a similar size (3.2 kb) whatever the salinity conditions. Thus, the transcriptional expression of the tilapia PRL-R strongly differs from the complex RNA pattern reported for the higher vertebrates PRL-R and provides an additional argument for the existence of a single PRL-R for both prolactin isoforms in this fish species.
Analysis of the 5'-upstream region of mouse P/Q-type Ca2+ channel alpha1A subunit gene for expression in pancreatic islet beta cells using transgenic mice and HIT-T15 cells
Authors: Takahashi E , Miyamoto N , Nagasu T
Published: December 2004

The omega-agatoxin-IVA-sensitive P/Q-type Ca(2+) channel plays a role in insulin release from the pancreatic islets of beta cells. To dissect the molecular mechanisms underlying beta cell expression of the P/Q-type channel, we characterized the 5'-upstream region of the mouse alpha(1A) subunit gene using transgenic mice and HIT insulinoma cells. The E. coli lacZ reporter gene was expressed in pancreatic acini and islets in transgenic mice carrying the 6.3 kb or 3.0 kb of the 5'-upstream region, although those with 1.5 kb or 0. 5 kb of the 5'-upstream region failed to show reporter expression on histological examination. As the expression of alpha(1A)subunit gene could not be detected in acini using RT-PCR analysis, the reporter expression in acini might have been ectopic expression. When linked to the placental alkaline phosphatase reporter gene to examine promoter activity for beta cell expression, the 6.3 kb and 3.0 kb fragment of the 5'-upstream region, but not the smaller 1.5 kb fragment, were able to drive reporter gene expression in HIT cells. The sequence between 3.0 and 1.5 kb upstream of the start codon enhanced thymidine kinase promoter activity in HIT cells, but not in fibroblast NIH3T3 cells. These results suggested that the beta cell-specific elements of the alpha(1A) subunit gene are likely to be located in the distal upstream region (-3021 to-1563) of the 5'-upstream sequence and that the 6.3 kb fragment of the 5'-upstream region alone might be a lack of a negative cis-regulatory element(s) to suppress the alpha(1A) subunit gene expression in acini.
Molecular cloning of equine transforming growth factor-beta1 reveals equine-specific amino acid substitutions in the mature peptide sequence
Authors: Nixon AJ , Brower-Toland BD , Sandell LJ
Published: December 2004

This study cloned and sequenced equine transforming growth factor (TGF)-beta1, yielding a unique nucleotide structure which predicted amino acid substitutions not seen in other mammalian species. The nucleotide sequence homology was 89% to bovine, 91% to man, 90% to ovine, and 86% to rat. Derived amino acid sequence comparison showed that the equine protein was unique, differing by two residues from man, cow, sheep, pig, and dog, and by three residues in the rat. Subsequent use of the cDNA clones to examine the expression of the TGF-beta1 gene in various tissues indicated predominant expression in adult spleen and kidney, with an age-related peak in cartilage expression at 12 months, followed by a decline as the animals matured. Northern blots showed that the predominant transcript sizes were 2.5 and 1.9 kb. More sensitive mRNA detection using PCR reaction showed peak cartilage TGF-beta mRNA levels in horses 0.7 and 1 year of age, with declining expression in older animals (2.5 and 5.5 years of age). In conclusion, although the primary nucleotide sequence of equine TGF-beta was relatively homologous to that of other species, the resulting amino acid sequence was unique to the horse, differing by two residues from the majority of mammalian sequences, where the peptide structure is identical. Expression of TGF-beta was particularly evident in spleen and kidney, and showed an age-related increase in expression in cartilage as the animals approached maturity and then a decline with progressive aging.
Structural characterization and effects on corticosteroid secretion of endothelin-1 and endothelin-3 from the frog Rana ridibunda
Authors: Wang Y , Remy-Jouet I , Delarue C , Letourneau M , Fournier A , Vaudry H , Conlon JM
Published: December 2004

ABSTRACT Despite the intensive study of endothelin (ET) in mammals, the primary structure and biological activity of the peptide is not known for any species of non-mammalian tetrapod. Extracts of the stomach and the liver of the European green frog Rana ridibunda contained ET-like immunoreactivity measured by RIA using an antiserum raised against human ET-1. The amino acid sequence of the peptide that was isolated in pure form from the stomach extract was identical to that of human ET-1 and the peptide purified from the liver extract was identical to human ET-3 except for a single amino acid substitution (Phe(4)-->Tyr). These observations demonstrate that the amino acid sequences of ET family peptides have been very strongly conserved during evolution of tetrapods and suggest that the pathway of post-translational processing of preproendothelin in the frog is similar to that in mammals. Both frog/human ET-1, frog ET-3 and human ET-3 produced a concentration-dependent increase in the production of corticosteroids from perifused slices of the frog interrenal gland. The maximum responses produced by the peptides (approximately 2-fold increase over basal levels for both corticosterone and aldosterone production) were not significantly different. The potency of ET-1 (-log EC(50)=9.81+/-0.01 (s.e.m.) for corticosterone and 9.52+/-0.29 for aldosterone production) was significantly (P<0.01) greater than that of frog ET-3 (-log EC(50)=8.13+/-1.6 for corticosterone and 8.15+/-0.33 for aldosterone production) but the potencies of frog ET-3 and human ET-3 (-log EC(50)=8.29+/-0.34 and 7.87+/-0.18) were not significantly different.
Expression of 11beta-hydroxylase and aldosterone synthase genes in the rat brain
Authors: MacKenzie SM , Clark CJ , Fraser R , Gomez-Sanchez CE , Connell JM , Davies E
Published: December 2004

The terminal stages of cortisol and aldosterone production in the human adrenal gland are catalysed by the enzymes 11beta-hydroxylase and aldosterone synthase, which are encoded by the CYP11B1 and CYP11B2 genes respectively. Recent studies have suggested that aldosterone and cortisol are also made in other tissues such as the brain, heart and vascular system and may play a role in cardiovascular homeostasis. The aim of this study was to confirm the presence of these enzymes and localise them precisely in the rat brain. Reverse transcription-polymerase chain reaction (RT-PCR)/Southern blotting confirmed transcription of CYP11B1 and CYP11B2 in whole brain and hypothalamus minces from Wistar-Kyoto rats. 11beta-Hydroxylase and aldosterone synthase were immunolocalised in paraffin-embedded rat adrenal and brain sections using mouse monoclonal antibodies. Negative controls utilised a mouse monoclonal antibody raised against a non-mammalian epitope. In the brain, 11beta-hydroxylase and aldosterone synthase were detected in the cerebellum, especially the Purkinje cells, as well as the hippocampus. The specificities of the 11beta-hydroxylase and aldosterone synthase antibodies were confirmed by positive immunostaining of the relevant regions of the adrenal cortex. This is the first direct evidence that steroid hydroxylases involved in the final stages of corticosteroid biosynthesis are present in specific regions of the central nervous system.
Rapid hormonal regulation of N-acetylglucosamine transferase I
Authors:   Bolander FF
Published: December 2004

Previous studies have shown that, in unstimulated mammary epithelial cells from virgin mice, prolactin receptors are retained intracellularly because of their incomplete N-glycosylation. Activation of the nitric oxide/cGMP pathway stimulates Nacetylglucosamine (NAG) transferase I activity, completion of terminal glycosylation, and redistribution of the receptors to the cell surface. In this study, it was shown that nitric oxide could stimulate the phosphorylation of NAG transferase I in intact cells and that the cGMP-dependent protein kinase (PKG) could directly phosphorylate the purified enzyme. Furthermore, this modification was associated with enhanced enzymatic activity. Conversely, this stimulation of activity was blocked in intact cells by coincubation with a PKG inhibitor and reversed in the immunoprecipitated enzyme by alkaline phosphatase treatment. Kinetic analysis revealed that this effect on enzyme activity was due to an increase in V(max) without any change in K(m). Therefore, it appears that the nitric oxide/cGMP pathway activates NAG transferase I via direct phosphorylation by PKG.
TSH controls Ref-1 nuclear translocation in thyroid cells
Authors: Tell G , Pellizzari L , Pucillo C , Puglisi F , Cesselli D , Kelley MR , Di Loreto C , Damante G
Published: December 2004

Ref-1 (called also APE) is a bifunctional protein playing a role in a large variety of cell functions. It is a major member of the DNA base excision repair system. Moreover, through reduction of cysteine residues, Ref-1 controls the activity of several transcription factors. It has been previously demonstrated that TSH up-regulates Ref-1 gene expression in thyroid cells. By using the rat FRTL-5 cell line, we demonstrate that TSH controls Ref-1 intracellular localization. Western blot experiments indicate that addition of TSH to the culture medium increases the Ref-1 cytoplasm-to-nucleus translocation. This phenomenon occurs at early times of TSH stimulation and is not dependent on protein neosynthesis. The Ref-1 cellular compartmentalization was also investigated in human thyroid tumors. A Ref-1 nuclear/cytoplasmic ratio difference between normal and cancerous thyroid tissues was observed. These results suggest that Ref-1 localization may have a critical role in the control of thyroid cell functions.
Opposite regulation of brain and C-type natriuretic peptides in the streptozotocin-diabetic cardiopathy
Authors: Walther T , Heringer-Walther S , Tschope R , Reinecke A , Schultheiss HP , Tschope C
Published: December 2004

C-type natriuretic peptide (CNP), a recent addition to the family of natriuretic peptides including atrial and brain natriuretic peptide (ANP, BNP), is believed to be an endothelium-derived vasodilator and to have an antimitotic effect. ANP and BNP concentrations are increased in conditions such as congestive heart failure, but cardiac CNP concentrations have not been investigated in this connection. Diabetes mellitus also involves myocardial dysfunctions without coronary artery disease or systemic hypertension. We therefore investigated the cardiac expression of CNP mRNA compared with that of BNP mRNA in streptozotocin (STZ)-diabetic rats. STZ- diabetic male Wistar rats (n=6) were studied in comparison with controls (n=6). The animals were characterised by their mean arterial blood pressure and plasma glucose concentrations. After extraction of total cardiac RNA, a specific cDNA probe of BNP was used for northern blot analysis, whereas myocardial CNP expression was analysed by an RNase-protection assay. Twelve weeks after diabetes was induced, the rats were normotensive (96.4+/-2.0 compared with 95.1+/-1.9 mmHg) and hyperglycaemic (615+/-61 compared with 165+/-21 mg/dl; P<0.001). Left ventricular pressure was significantly impaired (76.8+/-6.4 compared with 51.2+/-3.6 mmHg). STZ-diabetic rats had a 3.2-fold increase in cardiac BNP expression compared with controls. In contrast, cardiac CNP mRNA concentrations were decreased 2.6-fold. CNP seems to be downregulated like other peptides with antimitotic and vasodilator activities (nitric oxide, prostacyclin, kinins). This may contribute to cardiac dysfunction in diabetes mellitus and suggests that stimulation of CNP expression could provide cardiac protection in such cases.
The juxtamembrane but not the carboxyl-terminal domain of the insulin receptor mediates insulin's metabolic functions in primary adipocytes and cultured hepatoma cells
Authors: Paz K , Boura-Halfon S , Wyatt LS , LeRoith D , Zick Y
Published: December 2004

Insulin-stimulated signaling pathways are activated upon interactions between the intracellular domains of the receptor and its downstream effectors. Insulin receptor substrate proteins (IRS-1, -2, -3 and -4) are the best-studied substrates for the insulin receptor kinase (IRK). We have previously shown that IRS-1 and IRS-2 interact with the juxtamembrane (JM) but not with the carboxyl-terminal (CT) region of the insulin receptor (IR) in vitro. However, the precise role of these IR regions in mediating insulin's bioeffects is still unresolved. In the present work we made use of vaccinia virus as a vector for quantitative expression of the JM and CT domains within the cytoplasm of physiologically insulin-responsive primary rat adipocytes and rat hepatoma Fao cells. We could demonstrate that overexpression of either the JM or the CT domains did not inhibit either insulin binding or insulin-stimulated receptor autophosphorylation. In contrast, metabolic effects such as insulin-induced glucose utilization in adipocytes, and insulin-induced amino acid utilization in Fao hepatoma cells were inhibited (70-80%) in cells overexpressing the JM but not the CT domains of IR. The inhibitory effects of the overexpressed JM domain were accompanied by inhibition of insulin-stimulated IRS-1 phosphorylation, decreased IRS-1-associated PI3K activity, and decreased phosphorylation of the downstream effectors of PI3K, PKB and p70 S6K. Insulin-stimulated thymidine incorporation in Fao cells was also inhibited (40%) upon overexpression of the JM but not the CT region of IR. Our findings suggest that interactions between the JM region of IR and its downstream effectors are obligatory for insulin-stimulated metabolic functions in physiologically relevant insulin responsive cells. They also rule out the possibility that interaction of proteins, including PI3K, with the CT domain can provide an alternative pathway.
mRNA expression profiles for corticotrophin-releasing factor (CRF), urocortin, CRF receptors and CRF-binding protein in peripheral rat tissues
Authors: Baigent SM , Lowry PJ
Published: December 2004

The expression and activities of corticotrophin-releasing factor (CRF), urocortin (UCN), the CRF-binding protein (CRF-BP) and CRF receptors in rat brain have been well documented; however, information regarding their peripheral distributions remains incomplete. Given the multiple immunomodulatory effects of peripherally administered CRF and UCN and the high levels of CRF receptor type 2 (CRF-R2) mRNA and protein expressed in the heart, the lymphoid organs and heart have become targets for some of the latest CRF-related research. Here we demonstrate the presence of UCN mRNA in both the rat spleen and human Jurkat T-lymphoma cells using 3'-RACE (rapid amplication of cDNA ends) PCR. Following on from these initial results, we used semi-quantitative RT-PCR to carry out a comprehensive study assessing the relative amounts of CRF, UCN, CRF-R1, CRF-R2 and CRF-BP mRNAs in the brain, thymus, spleen and heart of normal, untreated rats. The rank orders of mRNA abundance in each of the tissue types were as follows: for CRF, brain>>thymus=spleen=heart; for UCN, heart>/=brain>thymus>spleen; for CRFR1, brain>>thymus>spleen (absent in heart); for CRF-R2, brain=heart>thymus>spleen; and CRF-BP was only detectable in the brain. We have provided evidence for the existence of CRF, UCN, CRF-R1 and CRF-R2 expression in resting immune cells, with UCN expression being particularly predominant in the rat thymus and human Jurkat cells. Additionally, the high levels of UCN mRNA detected in heart corresponded to the high expression of CRF-R2 mRNA, suggesting an important role for UCN/CRF-R2 coupling in this tissue.
Somatostatin receptor gene expression and inhibitory effects of octreotide on primary cultures of orbital fibroblasts from Graves' ophthalmopathy
Authors: Pasquali D , Vassallo P , Esposito D , Bonavolonta G , Bellastella A , Sinisi AA
Published: December 2004

To explore the mechanism underlying the effects of the somatostatin (SST) analogue octreotide in Graves' ophthalmopathy (GO), we investigated the expression of SST and of SST receptor (sst(1-5)) genes in primary cultures of fibroblasts established from retroorbital tissue of GO patients and of control subjects. We determined also SST specific binding sites by competitive binding of [(125)ITyr(11)]SST-14 and the effect of octreotide on cell growth, cAMP accumulation, Bcl-2 intracellular levels and apoptosis in GO fibroblast primary cultures. All primary cultures expressed the SST gene transcript and one or more ssts that have a high affinity for the two analogues (class 1 sst. The sst(2) transcript was found in nine, sst(3) in five and sst(5) in eight out of ten GO cell cultures. Sst(2) was detected in all six, and sst(3) in four out of the six control cell cultures. Sst(4) was absent from all samples, and sst(1) was found only in six out of the ten GO samples. SST-14 and octreotide inhibited the binding of [(125)I-Tyr(11)]SST-14 with a half-maximal inhibition of binding (IC(50)) of 0.80+/-0.37 and 33. 7+/- 33.1 nmol/l respectively in GO cell cultures, and with an IC(50) of 0.9 and 1.5 nmol/l in control cultures. Octreotide (10(-6) and 10(-7) M) significantly decreased (P<0.001) forskolin-induced but not basal cAMP accumulation; at both doses for 72 h it inhibited cell growth (20 and 55% respectively), and induced apoptosis (20 and 40%), and abolished Bcl-2 protein in cell lysates. In conclusion, SST and sst transcripts are expressed and functional in cultured retroorbital fibroblasts. The presence of class 1 sst in GO tissue and the inhibition exerted by octreotide on retroorbital cell growth and activity in vitro may account for the effects of SST analogue administration in vivo in GO.
Parathyroid hormone induces rat interstitial collagenase mRNA through Ets-1 facilitated by cyclic AMP response element-binding protein and Ca(2+)/calmodulin-dependent protein kinase II in osteoblastic cells
Authors: Quinn CO , Rajakumar RA , Agapova OA
Published: December 2004

Parathyroid hormone (PTH), a powerful bone-resorbing agent, is capable of stimulating interstitial collagenase (MMP-13) mRNA production in osteoblastic cells. In this study, a PEA3 consensus binding sequence (-80; AGGAAGT) in addition to a 'TRE-like' sequence (-89; CGACTCA) in the 5' upstream regulatory region of the rat MMP-13 gene were examined. In response to PTH, there was a time-dependent increase in binding of nuclear factors to an oligonucleotide containing the PEA3 region (-95 to -71). This increase in binding was first observed at 0.5 h, peaked at 4 h (7. 6-fold) then returned to basal levels by 24 h. Mutagenesis of the PEA3 site in a chloramphenicol acetyl transferase (CAT) construct containing 5' upstream regulatory sequence of the rat MMP-13 gene significantly decreased activation by PTH. PTH-mediated binding of nuclear factors to an oligonucleotide containing the mutant PEA3 sequence was decreased as compared with the wild type. Mutation or deletion of the TRE-like sequence affected basal as well as PTH-mediated induction of corresponding CAT constructs. Treatment with KN93, a Ca(2+)/calmodulin-dependent protein kinase II specific inhibitor, greatly reduced the amount of protein binding to the PEA3 region in response to PTH which correlated to a notable decrease in the amount of MMP-13 mRNA produced in response to PTH. Antibodies against Ets-1, cyclic AMP response element (CREB)-binding protein (CBP) and CREB were capable of supershifting proteins binding to the oligonucleotide containing the PEA3 region. These data suggest a possible co-operative interaction of factors binding to the PEA3 and TRE-like sequences and provide the first indication of a role for a calcium-mediated pathway in the PTH induction of MMP-13 mRNA in osteoblastic cells.
Molecular evolution of the growth hormone-releasing hormone/pituitary adenylate cyclase-activating polypeptide gene family. Functional implication in the regulation of growth hormone secretion
Authors: Montero M , Yon L , Kikuyama S , Dufour S , Vaudry H
Published: December 2004

Growth hormone-releasing hormone (GHRH) and pituitary adenylate cyclase-activating polypeptide (PACAP) belong to the same superfamily of regulatory neuropeptides and have both been characterized on the basis of their hypophysiotropic activities. This review describes the molecular evolution of the GHRH/PACAP gene family from urochordates to mammals and presents the hypothesis that the respective roles of GHRH and PACAP in the control of GH secretion are totally inverted in phylogenetically distant groups of vertebrates. In mammals, GHRH and PACAP originate from distinct precursors whereas, in all submammalian taxa investigated so far, including birds, amphibians and fish, a single precursor encompasses a GHRH-like peptide and PACAP. In mammals, GHRH-containing neurons are confined to the infundibular and dorsomedial nuclei of the hypothalamus while PACAP-producing neurons are widely distributed in hypothalamic and extrahypothalamic areas. In fish, both GHRH- and PACAP-immunoreactive neurons are restricted to the diencephalon and directly innervate the adenohypophysis. In mammals and birds, GHRH plays a predominant role in the control of GH secretion. In amphibians, both GHRH and PACAP are potent stimulators of GH release. In fish, PACAP strongly activates GH release whereas GHRH has little or no effect on GH secretion. The GHRH/PACAP family of peptides thus provides a unique model in which to investigate the structural and functional facets of evolution.
Characterization of glucagon-like peptide-1 receptor-binding determinants
Authors: Xiao Q , Jeng W , Wheeler MB
Published: December 2004

Glucagon-like peptide 1 (GLP-1) is a potent insulinotropic hormone currently under study as a therapeutic agent for type 2 diabetes. Since an understanding of the molecular mechanisms leading to high-affinity receptor (R) binding and activation may facilitate the development of more potent GLP-1R agonists, we have localized specific regions of GLP-1R required for binding. The purified N-terminal fragment (hereafter referred to as NT) of the GLP-1R produced in either insect (Sf9) or mammalian (COS-7) cells was shown to bind GLP-1. The physical interaction of NT with GLP-1 was first demonstrated by cross-linking ((125)I-GLP-1/NT complex band at approximately 28 kDa) and secondly by attachment to Ni(2+)-NTA beads. The GLP-1R NT protein attached to beads bound GLP-1, but with lower affinity (inhibitory concentration (IC(50)): 4.5 x 10(-7) M) than wild-type (WT) GLP-1R (IC(50): 5.2 x 10(-9)M). The low affinity of GLP-1R NT suggested that other receptor domains may contribute to GLP-1 binding. This was supported by studies using chimeric glucose-dependent insulinotropic polypeptide (GIP)/GLP-1 receptors. GIP(1-151)/GLP-1R, but not GIP(1-222)/GLP-1R, exhibited specific GLP-1 binding and GLP-1-induced cAMP production, suggesting that the region encompassing transmembrane (TM) domain 1 through to TM3 was required for binding. Since it was hypothesized that certain charged or polar amino acids in this region might be involved in binding, these residues (TM2-TM3) were analyzed by substitution mutagenesis. Five mutants (K197A, D198A, K202A, D215A, R227A) displayed remarkably reduced binding affinity. These studies indicate that the NT domain of the GLP-1R is able to bind GLP-1, but charged residues concentrated at the distal TM2/extracellular loop-1 (EC1) interface (K197, D198, K202) and in EC1 (D215 and R227) probably contribute to the binding determinants of the GLP-1R.
Thyroid hormone action in mitochondria
Authors: Wrutniak-Cabello C , Casas F , Cabello G
Published: December 2004

Triiodothyronine (T3) is considered a major regulator of mitochondrial activity. In this review, we show evidence of the existence of a direct T3 mitochondrial pathway, and try to clarify the respective importance of the nuclear and mitochondrial pathways for organelle activity. Numerous studies have reported short-term and delayed T3 stimulation of mitochondrial oxygen consumption. Convincing data indicate that an early influence occurs through an extra-nuclear mechanism insensitive to inhibitors of protein synthesis. Although it has been shown that diiodothyronines could actually be T3 mediators of this short-term influence, the detection of specific T3-binding sites, probably corresponding to a 28 kDa c-Erb Aalpha1 protein of the inner membrane, also supports a direct T3 influence. The more delayed influence of thyroid hormone upon mitochondrial respiration probably results from mechanisms elicited at the nuclear level, including changes in phospholipid turnover and stimulation of uncoupling protein expression, leading to an increased inner membrane proton leak. However, the involvement of a direct mitochondrial T3 pathway leading to a rapid stimulation of mitochondrial protein synthesis has to be considered. Both pathways are obviously involved in the T3 stimulation of mitochondrial genome transcription. First, a 43 kDa c-Erb Aalpha1 protein located in the mitochondrial matrix (p43), acting as a potent T3-dependent transcription factor of the mitochondrial genome, induces early stimulation of organelle transcription. In addition, T3 increases mitochondrial TFA expression, a mitochondrial transcription factor encoded by a nuclear gene. Similarly, the stimulation of mitochondriogenesis by thyroid hormone probably involves both pathways. In particular, the c-erb Aalpha gene simultaneously encodes a nuclear and a mitochondrial T3 receptor (p43), thus ensuring coordination of the expression of the mitochondrial genome and of nuclear genes encoding mitochondrial proteins. Recent studies concerning the physiological importance of the direct mitochondrial T3 pathway involving p43 led to the conclusion that it is not only involved in the regulation of fuel metabolism, but also in the regulation of cell differentiation. As the processes leading to or resulting from differentiation are energy-consuming, p43 coordination of metabolism and differentiation could be of significant importance in the regulation of development.