Hybridoma cells bearing monoclonal anti- body against. the j3-adrenergic ligand alprenolol were used as an immnunogen to raise. monoclonal anti-idiotypi antibodies. Of six anti-idiotypic antibodies, which inhibit.ligand binding, three were able to recognize IB-adrenergic receptors. One of them, mAb2B4, an IgM that could be amplified into ascites, binds to the fi-adrenergic catecholamine receptors of intact epidermoid A431 cells and precipitates receptors solubilized from plastna membranes by digitonin. This antibody identifies the 32-adrenergic receptor of A431 cells as a single. 55-kDa protein and stimulates adenylate cyclase activity. This stimula- tion is inhibited by the 18-adrenergic antagonist propranolol.

A high frequency of allelic loss affecting chromosome 8p and a minimal region of deletion at p21-22 have been previously reported in hepatocellular carcinoma (HCC), suggesting that at least one tumor suppressor gene is present in this region. In this study, we assessed whether the angiotensin II AT2 receptor interacting protein (ATIP)/mitochondrial tumor suppressor gene (MTUS1), a gene newly identified at position 8p22, may be a candidate tumor suppressor gene mutated in HCC. We searched for alterations in the 17 coding exons of ATIP/MTUS1 by means of denaturating high-performance liquid chromatography and sequencing, in 51 HCC tumors and 58 cell lines for which loss of heterozygosity status was known. Five major nucleotide substitutions were identified, all located in exons used by the ATIP3 transcript which is the only ATIP transcript variant expressed in liver. These nucleotide variations result in amino-acid substitution or deletion of conserved structural motifs (nuclear localisation signal, polyproline motif, leucine zipper) and also affect exonic splicing enhancer motifs and physiological splice sites, suggesting potential deleterious effects on ATIP3 function and/or expression.

The Mitochondrial Tumor suppressor 1 (MTUS1) gene is a newly identified candidate tumor suppressor gene at chromosomal position 8p22. We report here that MTUS1 encodes a family of proteins whose leader member (ATIP 1) was previously isolated in our laboratory as a novel interacting partner of the angiotensin II AT2 receptor involved in growth inhibition (Nouet, JBC 279: 28989-97, 2004). The MTUS1 gene contains 17 coding exons distributed over 112 kb of genomic DNA. Alternative exon usage generates three major transcripts (ATIP1, ATIP3 and ATIP4), each showing different tissue distribution. ATIP polypeptides are identical in their carboxy-terminal region carrying four coiled-coil domains. In their amino-terminal portion, ATIP polypeptides exhibit distinct motifs for localisation in the cytosol, nucleus or cell membrane, suggesting that MTUS1 gene products may be involved in a variety of intracellular functions in an AT2-dependent and independent manner. ATIP1 is ubiquitous and highly expressed in the brain. ATIP3 is the major transcript in tissues (prostate, bladder, breast, ovary, colon) corresponding to cancer types with frequent loss of heterozygosity at 8p22. Interestingly, ATIP4 is a brain-specific transcript highly abundant in the cerebellum and fetal brain. High evolutionary conservation of ATIP amino-acid sequences suggests important biological roles for this new family of proteins in tumor suppression and/or brain function. (c) 2006 Elsevier B.V. All rights reserved.

OBJECTIVESInterleukin-1 (IL-1) and interleukin-6 (IL-6) are determining factors in the immune and inflammatory responses to tumors cells. Experimental data suggest that interleukin-1 and interleukin-6 play important roles in the development and progression of breast cancer. We designed a broad study to investigate the susceptibility and prognostic implications of the genetic variation in IL-1alpha, IL-1beta and IL-6 in breast carcinoma.EXPERIMENTAL DESIGNWe used the polymerase chain reaction and restriction enzyme digestion to characterize the genetic variation of IL-1alpha, IL-1beta and IL-6 in 305, unrelated Tunisian patients with breast carcinoma and 200 healthy control subjects. Associations between the genetic markers and the clinicopathological parameters, the specific overall survival rate (OVS) of breast carcinoma and the disease free-survival rate (DFS) were assessed using univariate and multivariate analyses.RESULTSBoth IL-6 (-597) GA and IL-6 (-174) GC heterozygous genotypes were found to be significantly associated with breast carcinoma (OR = 1.59, p = 0.024 and OR = 1.61, p = 0.022 respectively). A highly significant association was found between the (+3954) T allele of IL1-B gene and the aggressive phenotype of breast carcinoma as defined by the high histological grade, axillary lymph node metastasis and large tumor size. The IL-1alpha (-889) TT homozygous genotype showed a significant association with reduced disease-free survival and/or overall survival rate. The IL-1beta (+3954) TT, IL-6 (-597) GG and IL-6 (-174) GG homozygous genotypes were found to be associated with reduced DFS but not with overall survival.CONCLUSIONSThe polymorphisms in the promoter region of the IL-6 gene may represent a marker for the increased risk of breast carcinoma. Genetic variations in IL-1alpha, IL-1beta and IL-6 may predict the clinical outcome of breast carcinoma.

Negative regulation of mitogenic pathways is a fundamental process that remains poorly characterized. The angiotensin II AT2 receptor is a rare example of a 7-transmembrane domain receptor that negatively cross-talks with receptor tyrosine kinases to inhibit cell growth. In the present study, we report the molecular cloning of a novel protein, ATIP1 (AT2-interacting protein), which interacts with the C-terminal tail of the AT2 receptor, but not with those of other receptors such as angiotensin AT1, bradykinin BK2, and adrenergic beta(2) receptor. ATIP1 defines a family of at least four members that possess the same domain of interaction with the AT2 receptor, contain a large coiled-coil region, and are able to dimerize. Ectopic expression of ATIP1 in eukaryotic cells leads to inhibition of insulin, basic fibroblast growth factor, and epidermal growth factor-induced ERK2 activation and DNA synthesis, and attenuates insulin receptor autophosphorylation, in the same way as the AT2 receptor. The inhibitory effect of ATIP1 requires expression, but not ligand activation, of the AT2 receptor and is further increased in the presence of Ang II, indicating that ATIP1 cooperates with AT2 to transinactivate receptor tyrosine kinases. Our findings therefore identify ATIP1 as a novel early component of growth inhibitory signaling cascade.

Blood levels of the satiety hormone leptin are directly correlated to fat stores in obese and lean people. Therefore, leptin resistance is the logical explanation for the phenomenon of common obesity. However, the important question of whether or not the intrinsic leptin activity could differ between obese and lean people has not been examined before. In the present study, serum leptin activity was measured by an in vitro assay of leptin signaling in a modified culture of HEK-293 cells. The system is based on activation of a luciferase reporter gene through a leptin receptor-dependent activation of the signal transducer and activator of transcription (STAT3). Serum samples from 20 obese and 20 non-obese individuals with leptin levels ranging from 3 to 75 ng/ml, as determined by radioimmunoassay (RIA), were used. A high correlation was observed for each serum sample between leptin RIA values and leptin activity in the bioassay. The results indicate that obesity in the 20 obese patients among the 40 individuals examined cannot be accounted for by alterations in leptin activity in our assay. The assay system provides a tool to screen for possible rare cases exhibiting alteration in leptin activity either due to a change in leptin itself or through interaction with other serum factors.

In addition to their role in inflammation, cytokines like TNFα have been reported to regulate the adipose tissue function suggesting a role for these soluble mediators in metabolism. However, it is not known whether adipocytes have the capacity to secrete chemokines, a group of low molecular weight inflammatory mediators that control leukocyte migration into tissues. Here we show that primary cultures of human preadipocytes constitutively produce three chemokines, interleukin-8 (IL-8), macrophage inflammatory protein-1α (MIP-1α) and monocyte chemotactic protein-1 (MCP-1), while their level of expression is low in mature adipocytes. Upon TNFα treatment, the expression of all the three chemokines is upregulated in adipocytes differentiated in vitro. In addition, we describe the presence of seven different chemokine receptors, mainly in mature adipocytes, both in vitro and in human fat tissue sections. Prolonged stimulation of cultured human adipocytes with exogenous chemokines leads to a decrease in lipid content in association with the downregulation of PPARγ mRNA expression. Moreover, chemokines positively control the secretion of leptin, a hormone that regulates appetite, by a post-transcriptional mechanism. These findings reveal a new role for chemokines in the regulation of adipose tissue and suggest a novel therapeutic basis for the treatment of obesity, diabetes and cachexia.

SM-11044 is the only β-adrenergic agonist that inhibits guinea pig eosinophil chemotaxis and induces relaxation of depolarized rat colon tonus. We have previously reported the purification of a 34 kDa photoaffinity-labeled SM-11044 binding protein (SMBP) from rat colon that may mediate the biological effects of the ligand and that differs from all known monoamine receptors (Sugasawa et al., J. Biol. Chem. 272 (1997) 21244). The present report describes partial amino acid sequence of rat SMBP and molecular cloning of corresponding human SMBP (hSMBP) cDNA. This cDNA encodes a 588 amino acid residue polypeptide comprising a signal peptide, a long hydrophilic amino-terminal region, and a highly hydrophobic C-terminal portion organized into nine putative transmembrane domains. The sequence and structure of hSMBP shows homology to members of a new transmembrane protein 9 superfamily (TM9SF). Comparison of hSMBP with related protein sequences from yeast, plant and human revealed two subgroups within TM9SF. The members of these groups differ in length and have characteristic amino acid sequence motifs in their amino-terminal portion. Northern blot analysis revealed two major SMBP mRNAs, at 3.4 and 3.8 kb, that were present in all the human tissues examined. Western blot experiments detected SMBP as a 70 kDa protein that may be further cleaved into an active 34 kDa N-terminal polypeptide. Stable Chinese Hamster Ovary cell transfectants expressing hSMBP cDNA displayed specific binding of [125I]iodocyanopindolol that was displaced by SM-11044 in a dose-dependent manner. Thus, SMBP is the first member of TM9SF with functional ligand binding properties, suggesting that some of these integral membrane proteins may function as channels, small molecule transporters or receptors.

Aims/hypothesis. Adipose tissue-derived tumour necrosis factor-α (TNF-α) has been implicated in the insulin resistance observed in animal models of obesity. Moreover, TNF-α has inhibitory effects on adipocyte differentiation. Glucocorticoids play important roles in the regulation of insulin sensitivity and adipose tissue distribution. We therefore studied the effect of dexamethasone on TNF-α expression and adipocyte differentiation in human PAZ6 cells. Methods. The expression of TNF-α and adipocyte differentiation markers was assessed by reverse-transcription polymerase chain reaction in PAZ6 cells. Results. In cells cultured for 15 days in the presence of dexamethasone, adipocyte differentiation marker expression was higher and TNF-α expression was lower than in cells cultured in the absence of dexamethasone. The presence of dexamethasone was necessary during the whole period of differentiation because removal of dexamethasone during the second week resulted in poorly differentiated adipocytes that express higher levels of TNF-α.

BACKGROUND : Tumor necrosis factor-α (TNF-α) expression is increased in adipose tissue of both rodent models of obesity and obese humans. It has therefore been considered as a candidate gene for obesity. Several studies have indeed shown statistical evidence of linkage between obesity and the chromosomal region encompassing the TNF-α gene, suggesting that TNF-α and/or a nearby gene (eg hsp70 gene) is involved in the onset and progression of weight gain. We designed a case-controlled study to investigate the potential association of polymorphism of the TNF-α and that of a stress protein (hsp70-2) with obesity. METHODS : We used the polymerase chain reaction and restriction enzyme digestion to characterize the variation of the TNF-α promoter region and that of the hsp70-2 gene in 343 unrelated Tunisian patients with obesity and 174 healthy control subjects. RESULTS : Analysis of the -308 TNF-α polymorphism in patients with obesity and in control subjects did not reveal an association between TNF-α alleles and obesity. In contrast, polymorphism analysis of the hsp70-2 gene in patients with obesity demonstrated highly significant differences in genotypic distribution of this bi-allelic locus compared to the control subject group. Homozygosity for one hsp70-2 allele was highly associated with obesity ( r 2 =7.12; P <10 −6 ). CONCLUSION : Tunisian persons carrying the P2/P2 genotype of the hsp70-2 gene may have an increased risk of obesity. International Journal of Obesity (2001) 25, 462–466

G protein-coupled receptors (GPCRs) are among the most important targets for pharmacological compounds used in humans. During the last 10 years, more than 1000 genes coding for GPCRs, representing approx 0.1% of the human genome, have been identified, of which at least 200 have been demonstrated to correspond to active receptors. Cloning of receptor cDNAs allowed expression in heterologous cell systems. Among the available expression systems, considerable interest has focused on microorganisms, such as Escherichia coli (E. coli), that provide a reproducible and inexpensive source of receptor, combined with ease of use, low background binding, and the absence of endogenous GPCR. All these characteristics are prerequisites for the successful application of GPCRs in high-throughput screening assays for the discovery of new drugs (1).

The present study demonstrates negative intracellular cross-talk between angiotensin II type 2 (AT2) and insulin receptors. AT2 receptor stimulation leads to inhibition of insulin-induced extracellular signal-regulated protein kinase (ERK2) activity and cell proliferation in transfected Chinese hamster ovary (CHO-hAT2) cells. We show that AT2 receptor interferes at the initial step of insulin signaling cascade, by impairing tyrosine phosphorylation of the insulin receptor (IR) beta-chain. AT2-mediated inhibition of IR phosphorylation is insensitive to pertussis toxin and is also detected in neuroblastoma N1E-115 and pancreatic acinar AR42J cells that express endogenous receptors. We present evidence that AT2 receptor inhibits the autophosphorylating tyrosine kinase activity of IR, with no significant effect on insulin binding properties. AT2-mediated inactivation of IR does not mainly involve tyrosine dephosphorylation by vanadate-sensitive tyrosine phosphatases nor serine/threonine phosphorylation by protein kinase C. As a consequence of IR inactivation, AT2 receptor inhibits tyrosine phosphorylation of insulin receptor substrate-1 (IRS-1) and signal-regulatory protein (SIRPalpha1) and prevents subsequent association of both IRS-1 and SIRPalpha1 with Src homology 2 (SH2)-containing tyrosine phosphatase SHP-2. Our results thus demonstrate functional trans-inactivation of IR kinase by G protein-coupled AT2 receptor, illustrating a novel mode of negative communication between two families of membrane receptors.

The citrus phospholipid hydroperoxide glutathione peroxidase (cit-PHGPx) was the first plant peroxidase demonstrated to exhibit PHGPx-specific enzymatic activity, although it was 500-fold weaker than that of the pig heart analog. This relatively low activity is accounted for the catalytic residue of cit-PHGPx, which was found to be cysteine and not the rare selenocysteine (Sec) present in animal enzymes. Sec incorporation into proteins is encoded by a UGA codon, usually a STOP codon, which, in prokaryotes, is suppressed by an adjacent downstream mRNA stem-loop structure, the Sec insertion sequence (SECIS). By performing appropriate nucleotide substitutions into the gene encoding cit-PHGPx, we introduced bacterial-type SECIS elements that afforded the substitution of the catalytic Cys(41) by Sec, as established by mass spectrometry, while preserving the functional integrity of the peroxidase. The recombinant enzyme, whose synthesis is selenium-dependent, displayed a 4-fold enhanced peroxidase activity as compared with the Cys-containing analog, thus confirming the higher catalytic power of Sec compared with Cys in cit-PHGPx active site. The study led also to refinement of the minimal sequence requirements of the bacterial-type SECIS, and, for the first time, to the heterologous expression in Escherichia coli of a eukaryotic selenoprotein containing a SECIS in its open reading frame.

Abstract: We have previously shown that the mitogenic effect of endothelin‐1 (ET‐1) in primary astrocytes is dependent on activation of both extracellular signal‐regulated kinase (ERK)‐ and cytoskeleton (CSK)‐dependent pathways. In this study, we evaluated the contribution of each of these pathways to the expression and activation of proteins mediating cell cycle progression. Our results suggest that ET‐1‐induced expression of cyclins D1 and D3 is dependent on the ERK‐ and CSK‐dependent pathways, respectively; moreover, a decrease in the levels of the cyclin‐dependent kinase inhibitor (CKI) p27 was observed as a consequence of ERK activation. Expression of both cyclins D1 and D3 together with a decrease in the p27 levels are essential for retinoblastoma protein (pRB) phosphorylation and cyclin A expression. Furthermore, the molecular events responsible for cell—cell contact inhibition of astrocyte proliferation were found to be independent of the mitogenic pathways leading to D‐type cyclin expression. Cell growth arrest in confluent astrocytes was found to be correlated with increased expression of CKI p21, resulting in inhibition of D‐type cyclin‐associated pRB phosphorylation and cyclin A expression. Taken together, these results indicate that cyclins D1 and D3, which constitute the key mediators of the proliferative response of primary astrocytes to ET‐1, are regulated by distinct signaling pathways.

Introduction - Structure and Function of the ss3-Adrenoreceptor, A.D. Strosberg and C.C. Gerhardt Regulation of the ss3-Adrenoreceptor Signalling Efficacy, M. Bouvier Using Transgenic and Gene Knockout Techniques to Assess ss3-Adrenoreceptor Function, B.B. Lowell, V.S. Susulic, D. Grujic, and M. Ito ss3-Adrenoreceptor Ligands and the Pharmacology of ss3-Adrenoreceptor, J.R.S. Arch The Native Human ss3-Adrenoreceptor, P. Arner and F. Lonnqvist ss3-Adrenoreceptor-Mediated Responses in Heart and Vessels, M. Lafontan, D. Langin, J. Galitzky, M. Berlan, C. Gauthier, and G. Tavernier ss3-Adrenoreceptors in Brown and White Adipocytes: Roles in Thermogenesis and Energy Balance, J. Himms-Hagen The Putative "ss4"-Adrenoreceptor and Other Atypical ss-Adrenoreceptors, A.D. Srosberg, F. Lonnqvist, J.R.S. Arch, M. Bouvier, J. Himms-Hagen, M. Lafontan, D. Langin, J. Galitzky, M. Berlan, C. Gauthier, G. Tavernier, B.B. Lowell, V.S. Susulic, D. Grujic, M. Ito, A.D. Strosberg