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Hepatitis C virus (HCV) infects over 130 million people causing a worldwide epidemic of liver cirrhosis and hepatocellular-carcinoma. Because current HCV treatments are only partially effective, molecular mechanisms involved in HCV propagation are actively being pursued as possible drug targets. Here, we report on a new macromolecular interaction between the HCV capsid core protein and the helicase portion of HCV non-structural protein 3 (NS3h), confirmed by four different biochemical methods. The protease portion of NS3 is not required. Interaction between the two proteins could be disrupted by two types of specific inhibitors of core dimerization, the small molecule SL201 and core106, a C-terminally truncated core protein. Cross-linking experiments suggest that the physical interaction with NS3h is probably driven by core oligomerization. Moreover, SL201 blocks the production of infectious virus, but not the production of a subgenomic HCV replicon by hepatoma cells. Time-of-addition experiments confirm that SL201 has no effect on entry of the virus. These data underline the essential role of core as a key organizer of HCV particle assembly, confirm the importance of oligomerization, reveal the interaction with viral helicase and support a new molecular understanding of the formation of the viral particle at the level of the lipid droplets, before its migration to the site of release and budding.

Binding of hepatitis C virus (HCV) RNA to core, the capsid protein, results in the formation of the nucleocapsid, the first step in the assembly of the viral particle. A novel assay was developed to discover small molecule inhibitors of core dimerization. This assay is based on time-resolved fluorescence resonance energy transfer (TR-FRET) between anti-tag antibodies labeled with either europium cryptate (Eu) or allophycocyanin (XL-665). The N-terminal 106-residue portion of core protein (core106) was tagged with either glutathione-S-transferase (GST) or a Flag peptide. Tag-free core106 was selected as the reference inhibitor. The assay was used to screen the library of pharmacologically active compounds (LOPAC) consisting of 1,280 compounds and a 2,240-compound library from the Center for Chemical Methodology and Library Development at Boston University (CMLD-BU). Ten of the 28 hits from the primary TR-FRET run were confirmed in a secondary amplified luminescent proximity homogeneous assay (ALPHA screen). One hit was further characterized by dose-response analysis yielding an IC(50) of 9.3 microM. This 513 Da compound was shown to inhibit HCV production in cultured hepatoma cells.

Background Functional brown adipose tissue (BAT), involved in energy expenditure, has recently been detected in substantial amounts in adults. Formerly overlooked BAT has now become an attractive anti-obesity target. Methods and results Molecular characterization of human brown and white adipocytes, using a myriad of techniques including high-throughput RNA sequencing and functional assays, showed that PAZ6 and SW872 cells exhibit classical molecular and phenotypic markers of brown and white adipocytes, respectively. However, the pre-adipocyte cell line SGBS presents a versatile phenotype. A transit expression of classical brown markers such as UCP1 and PPARγ peaked and declined at day 28 post-differentiation initiation. Conversely, white adipocyte markers, including Tcf21, showed reciprocal behavior. Interestingly, leptin levels peaked at day 28 whereas the highest adiponectin mRNA levels were detected at day 14 of differentiation. Phenotypic analysis of the abundance and shape of lipid droplets were consistent with the molecular patterns. Accordingly, the oxidative capacity of SGBS adipocytes peaked on differentiation day 14 and declined progressively towards differentiation day 28. Conclusions Our studies have unveiled a new phenotype of human adipocytes, providing a tool to identify molecular gene expression patterns and pathways involved in the conversion between white and brown adipocytes. Electronic supplementary material The online version of this article (doi:10.1186/s12967-015-0480-6) contains supplementary material, which is available to authorized users.

Leptin receptor (OB-R) splice variants either encode proteins with different 3' cytoplasmic domains or have different 5' untranslated regions (UTR), indicative of dual promoters. The B219/OB-R promoter transcribes only OB-R transcripts, whereas the OB-R/GRP promoter initiates transcription of both OB-R and another protein of unknown function, called the leptin receptor gene-related protein (OB-RGRP). We compared expression of B219/OB-R 5'-UTR and OB-RGRP mRNAs by in situ hybridization. We thus assessed, by inference, the contributions of the two promoters to the leptin receptor transcript pool, in murine brain or in placenta, a tissue with abundant leptin receptor mRNA. Expression of B219/OB-R 5'-UTR mRNA (and thus by inference B219/OB-R promoter activity) in brain was similar in both distribution and relative intensity to OB-R mRNA. OB-RGRP mRNA (and thus by inference OB-R/GRP promoter activity) was widely distributed in murine brain, with elevated expression in the hypothalamic regions that express the leptin receptor mRNA, including the paraventricular nucleus. B219/OB-R 5'-UTR mRNA, but not OB-RGRP mRNA, was upregulated in hypothalamus of obese ob/ob mice. In placenta, B219/OB-R 5'-UTR mRNA was restricted to the maternal interface, and transcription of both long and short leptin receptor splice variants in the main body of the tissue thus proceeds via the OB-R/GRP promoter, strongly indicative of tissue-specific promoter usage.

Several reports have demonstrated that the pineal hormone, melatonin, plays an important role in body mass regulation in mammals. To date, however, the target tissues and relevant biochemical mechanisms involved remain uncharacterized. As adipose tissue is the principal site of energy storage in the body, we investigated whether melatonin could also act on this tissue. Semiquantitative RT-PCR analysis revealed the expression of MT1 and MT2 melatonin receptor mRNAs in the human brown adipose cell line, PAZ6, as well as in human brown and white adipose tissue. Binding analysis with 2-[(125)I]iodomelatonin ((125)I-Mel) revealed the presence of a single, high affinity binding site in PAZ6 adipocytes with a binding capacity of 7.46 +/- 1.58 fmol/mg protein and a K(d) of 457 +/- 5 pM. Both melatonin and the MT2 receptor-selective antagonist, 4-phenyl-2-propionamidotetraline, competed with 2-[(125)I]iodomelatonin binding, with respective K(i) values of 3 x 10(-11) and 1.5 x 10(-11) M. Functional expression of melatonin receptors in PAZ6 adipocytes was indicated by the melatonin-induced, dose-dependent inhibition of forskolin-stimulated cAMP levels and basal cGMP levels with IC(50) values of 2 x 10(-9) and 3 x 10(-10) M, respectively. Modulation of the cGMP pathway by melatonin further supports functional expression of MT2 receptors, as this pathway was shown to be specific for that subtype in humans. In addition, long-term melatonin treatment of PAZ6 adipocytes was found to decrease the expression of the glucose transporter Glut4 and glucose uptake, an important parameter of adipocyte metabolism. These results suggest that melatonin may act directly at MT2 receptors on human brown adipocytes to regulate adipocyte physiology.

In addition to their role in inflammation, cytokines like TNFalpha 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-1alpha (MIP-1alpha) and monocyte chemotactic protein-1 (MCP-1), while their level of expression is low in mature adipocytes. Upon TNFalpha 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 PPARgamma 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.

Adipose tissue-derived tumour necrosis factor-alpha (TNF-alpha) has been implicated in the insulin resistance observed in animal models of obesity. Moreover, TNF-alpha 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-alpha expression and adipocyte differentiation in human PAZ6 cells.The expression of TNF-alpha and adipocyte differentiation markers was assessed by reverse-transcription polymerase chain reaction in PAZ6 cells.In cells cultured for 15 days in the presence of dexamethasone, adipocyte differentiation marker expression was higher and TNF-alpha 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-alpha. Dexamethasone also reduced TNF-alpha expression during early stages of differentiation. The use of a TNF-alpha-neutralising antibody showed, however, that endogenously-produced TNF-alpha did not play an important part in the control of PAZ6 cell differentiation. During early stages of adipocyte differentiation, dexamethasone induced the expression of the transcription factors PPAR gamma (peroxisome proliferator activated receptor gamma) and C/EBP alpha (CCAAT/enhancer binding protein alpha) while inhibiting the expression of the inhibitor of DNA binding Id2.The effect of dexamethasone on human adipocyte differentiation is not mediated by reduction of TNF-alpha expression but more likely by regulation of the expression of nuclear factors such as PPAR gamma, CEBP alpha and Id2.

Background: We previously localized β3-adrenergic receptors immunohistochemically in human gastrointestinal smooth muscle and incidently found a population of human pancreatic islet cells and duodenal epithelial neuroendocrine cells that also expressed β3-adrenergic receptors. Aim: To identify the nature of the islet and duodenal cells that stained positive for β3-adrenergic receptors. Methods: Paraffin sections of human pancreas and duodenum and Chinese hamster ovary cells transfected with the human β3-adrenergic receptor were immuno-stained for β3-adrenergic receptors using an affinity-purified rabbit polyclonal antibody (anti-P12) raised against a 15 amino acid sequence (P12) of the human receptor. Immunohistochemical staining for the receptor was carried out in the presence and absence of P12 peptide and both somatostatin 14 and 18 peptides. β3-adrenergic receptor-stained sections were also double-immunostained with anti-insulin, -glucagon, -somatostatin and -pancreatic polypeptide antibodies. Results: A subpopulation of human pancreatic islet cells and duodenal epithelial cells expressed positive cytoplasmic β3-adrenergic receptor immunostaining. Using distribution and double-staining techniques, these cells were found to be somatostatin-positive D cells but not A or B cells. The positive staining of D cells with anti-P12 antibody was inhibited by prior incubation of the antibody with P12 peptide but not somatostatin-14 or -28 peptides. Pancreatic vascular smooth muscle and duodenal vascular and non-vascular smooth muscle also stained with anti-P12 antibody. Transfected Chinese hamster ovary cells showed positive membrane staining. Conclusion: We have identified a population of neuroendocrine cells in the human pancreas and duodenum that express β3-adrenergic receptors. These cells appear to be somatostatin D cells.

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.

To investigate the relationship between the polymorphisms of the beta3-AR (Trp64Arg), UCP1 (A--G) and LPL (HindIII and PvuII) loci and the metabolic complications associated with obesity in a Turkish population.271 unrelated individuals of Turkish origin including obese (body mass index, BMI30 kg¿m2) and lean (BMIor =25 kg¿m2) subjects.Anthropometric (weight, height and blood pressure) and metabolic measurements (plasma levels of glucose, cholesterol and triglycerides), and determination of beta3-AR, UCP1 and LPL genotypes by polymerase chain reaction followed by enzymatic digestion.The distributions of genotypes for each candidate gene (beta3-AR, UCP1 and LPL) were similar between the obese and the lean subjects. The Arg64 allele of the beta3-AR gene was absent from massively obese men. GG carriers of the A--G variant of the UCP1 gene showed BMI-associated increases of cholesterol levels which were more marked than both AA (P=0.027) and AG (P=0.039) carriers. Obese P+ carriers of the LPL PvuII variant had significantly higher levels of glucose than non-carriers (P=0.011), whereas obese P+P+ carriers did not have significantly different levels of triglycerides than non-carriers (P=0.087). Moreover, carriers of both alleles (GP+) had higher levels of glucose than non-carriers (P=0.048), but did not have significantly different levels of triglycerides than non-carriers (P=0.125). However, the BMI-associated increase of triglycerides of P+G carriers was significantly more marked than that of P+ carriers (P=0.0085).Our data support the idea that alleles of specific genes (UCP1, LPL and beta3-AR) might play a role in the development of certain metabolic complications of obesity and might have additive effects when combined with each other (as in the case of UCP1 and LPL). International Journal of Obesity (2000)24, 93-100

Aims/hypothesis. Thiazolidinediones, a new class of insulin sensitizers, up-regulate the expression of uncoupling protein 2 in rodent adipocytes. It is not known, however, whether thiazolidinediones influence uncoupling protein 2 expression in human adipocytes. We therefore investigated the effect of these drugs on uncoupling protein 2 expression in the recently immortalized human PAZ6 adipocyte cell line. Methods. Immortalized human PAZ6 preadipocytes were differentiated into adipocytes in the presence or absence of thiazolidinediones. The effect of the drugs on uncoupling protein 2 expression and adipocyte differentiation was measured by reverse transcription-polymerase chain reaction of mRNA of uncoupling protein 2 and of five adipocyte differentiation markers. Results. When cells were differentiated 15 days in the presence of thiazolidinediones, uncoupling protein 2 expression was 2.1-fold higher than in the absence of the drugs. The expression of five adipocyte differentiation markers was, however, also increased by thiazolidinediones. Short-term incubation for 4 and 24 h with thiazolidinediones increased uncoupling protein 2 expression 1.35-fold and 2.3-fold, respectively. The expression of adipocyte markers studied in parallel was also augmented. Conclusion/interpretation. Thiazolidinediones rapidly increase the expression of uncoupling protein 2 in human PAZ6 adipocytes but the increase of uncoupling protein 2 expression is always associated with an augmentation of the expression of all adipocyte markers studied in parallel. This indicates that the effect of thiazolidinediones on uncoupling protein 2 mRNA reflects a general increase in adipocyte differentiation rather than a specific augmentation of uncoupling protein 2 gene expression. [Diabetologia (1999) 42: 527–533]

Objectives: a) To investigate the functional consequences of sepsis on the β-adrenergic signal transduction in human circulating lymphocytes; b) to appreciate sepsis-associated catecholamine and cytokine release. Design: Experimental, comparative study. Setting: Research laboratory in a university hospital. Subjects: Healthy controls (n = 10); critically ill patients who were not septic (n = 7); septic patients with severe sepsis or septic shock (n = 11). Measurements and main results: Experiments were carried out using freshly isolated peripheral blood mononuclear cells (PBMC). We measured β-adrenergic receptor (βAR) number and affinity, and intracellular cAMP content at baseline and after the pharmacological stimulation of each component of the β -adrenergic complex: βAR with isoproterenol, Gs-protein with sodium fluoride (NaF), adenylate cyclase with forskolin. Catecholamine (adrenaline, noradrenaline) and cytokine (TNFα, IL-1α, IL-1β, IL-6) serum levels were measured. In both septic and non-septic patients we observed a similar 40 % down-regulation of βARs compared to controls, and a reduced basal and isoproterenol-stimulated cAMP accumulation (p < 0.05). The cAMP production elicited by NaF or forskolin was lower in septic patients than in the controls (p < 0.01). Forskolin-stimulated cAMP accumulation was significantly lower in septic patients than it was in non-septic ones (p < 0.001). Catecholamine serum concentrations were increased in the two patient groups without any significant difference. Elevated cytokine serum levels were detected in 45 % of the septic patients (versus 14 % of non-septic patients p < 0.05). Conclusions: Patients presenting with severe sepsis or septic shock have extended postreceptor defects of the β-adrenergic signal transduction. This finding suggests a heterologous desensitization of adenylate cyclase stimulation.

A citrus salt-stress associated protein (Cit-SAP), partially purified from citrus cultured cells, was previously identified as the first plant phospholipid hydroperoxide glutathione peroxidase (PHGPx). The nucleotide sequence of its isolated gene (csa) revealed that a TGT, known as codon for Cys, encodes the presumed catalytic residue 41 in the polypeptide chain of Cit-SAP. In animals, a TGA encodes the rare amino acid selenocysteine (Sec) as the catalytic residue of the analogous enzyme. It is of interest to establish whether the TGT codon for this catalytic residue in the plant enzyme is indeed translated to Cys and not to Sec, and to demonstrate the effect of such a change, if it exists, on the nature of the enzymatic activity of the plant enzyme as compared to that of the animal. In the present study, we have purified for the first time, by affinity chromatography, enzymatically active citrus PHGPx from recombinant Escherichia coli bearing the csa gene. Tryptic digestion of the purified enzyme followed by HPLC afforded the isolation of a peptide which contains residue 41, and its sequence analysis revealed that this residue is indeed a Cys, and not Sec. The enzymatic activity and specificity of the recombinant Cit-SAP was found to be similar to that observed before for the partially purified plant enzyme. However, the rate of this activity was much lower towards phospholipid hydroperoxides, and none towards hydrogen peroxide, as compared to that of the animal analogue. It is therefore suggested that the presence of a Cys, and not Sec, as the catalytic residue in the plant enzyme, affects its enzymatic activity and may determine a different biological role for the plant PHGPx from that of the animal.

Background: Activation of human and non-human colonic β(beta)3-adrenoceptors causes smooth muscle relaxation. β3-Adrenoceptor agonists protect against experimental indomethacin-induced jejunal ulceration. The mechanism of protection may involve spasmolytic/vasodilatory agonist activity. The precise localization of β3-adrenoceptors in the human gut is not known. Aim: To localize the β3-adrenoceptor within the human gastrointestinal tract using the immunohistochemical technique. Methods: Human β3-adrenoceptors were immuno-localized in paraffin sections of human oesophagus (OS), stomach (ST), duodenum (DU), ileum (IL), sigmoid colon (SC), rectum (R) and gall-bladder (GB) using the rabbit polyclonal antibody anti-P12. Staining was graded +++, ++, + and 0. Immunostaining of SC was also done with pre-incubation of anti-P12 with P12 peptide. Western blotting of anti-P12 on human and murine IL and SC isolated membrane proteins was performed. Results: All epithelia, vascular endothelial cells and ganglia scored 0. Smooth muscle of the vasculature, muscularis propria, muscularis mucosae and mucosa was graded, respectively, as follows; OS (+++, +++, +++,-), ST (++, +++, ++, ++), DU (++, +++, +++, +), IL (++, ++, ++, +), SC (+++, ++, ++, ++), R (++, ++, +, +), GB (+++, +++, –, 0). Pre-incubation of anti-P12 with P12 peptide almost abolished SC smooth muscle positivity. Western blot analysis using anti-P12 on human, but not murine, IL and SC membrane proteins revealed a single 55 kDa band, a size consistent with the predicted size of a partially glycosylated form of the human β3-adrenoceptor. Conclusions: This immunohistochemical study has localized the β3-adrenoceptor to vascular and non-vascular smooth muscle in the human gastrointestinal tract. These findings support a role for the β3-adrenoceptor in the control of blood flow and motility in the human gastrointestinal tract.

Angiotensin II type 2 (AT2) receptors are involved in the inhibition of cell proliferation as well as in apoptosis and neuronal differentiation, through intracellular signalling pathways that remain poorly defined. The present study examines the effect of AT2-receptor stimulation on growth-factor-induced pathways leading to the activation of mitogen-activated protein (MAP) kinases. In N1E-115 neuroblastoma cells, AT2 receptors inhibit the activity of MAP kinases induced by serum as well as by epidermal growth factor. The inhibitory effect of angiotensin II (Ang II) is rapid and transient, and affects both ERK1 and ERK2 (extracellular signal-related protein kinase) isoforms of the enzyme. AT2-mediated MAP kinase inactivation is not sensitive to pertussis toxin or okadaic acid, but involves a vanadate-sensitive protein tyrosine phosphatase (PTP). Expression of MAP kinase phosphatase-1 (MKP-1) is not significantly modified upon AT2-receptor activation, and insensitivity to actinomycin D also rules out transcriptional induction of other MKPs as a possible mechanism for AT2-mediated inactivation of MAP kinases. In addition, we report here that both in N1E-115 cells and in Chinese hamster ovary cells expressing recombinant human AT2 receptors, Ang II rapidly stimulates the catalytic activity of SHP-1, a soluble PTP that has been implicated in termination of signalling by cytokine and growth-factor receptors. These findings thus demonstrate functional negative cross-talk between heptahelical AT2 receptors and receptor tyrosine kinases, and suggest that SHP-1 tyrosine phosphatase is an early transducer of the AT2 receptor signalling pathway.

Support for our work comes mostly from the Centre National de la Recherche Scientifique, the Institut National de la Sante et de la Recherche Medicale, the University of Paris VII, and the Ministry for Research and Technology. We are also grateful for help from the Ligue Nationale contre le Cancer, the Fondation pour la Recherche Medicale Francaise and last but not least, the Association pour la Recherche contre le Cancer.

▪ Abstract The β3 subtype of adrenaline and noradrenaline receptors has now been extensively characterized at the structural and functional levels. Ligand binding and adenylyl cyclase activation studies helped define a β-adrenergic profile that is quite distinct from that of the β1-and β2-adrenergic receptors, but strongly reminiscent of most of the “atypical” responses reported in earlier pharmacologic studies. Human, other large mammal, and rodent receptors share most of the characteristic β3 properties, although obvious species-specific differences have been identified. Recently, the incidence of a naturally occurring variant of the human β3-adrenergic receptor was shown to be correlated with hereditary obesity in Pima Indians and in Japanese individuals, and in Western obese patients with increased dynamic capacity to add on weight and develop non–insulin-dependent diabetes mellitus (NIDDM). A mild weight increase was also shown to develop in female, but not male, mice in which the β3 receptor gene was disrupted. Taken together, these results now provide a consistent picture of an important role of the β3-adrenoceptor in the regulation of lipid metabolism and as an obvious target for drugs to treat some forms of obesity.

Three beta-adrenergic receptor subtypes are now known to be functionally expressed in mammals. All three belong to the R7G family of receptors coupled to G-proteins, and characterized by an extracellular glycosylated N-terminal and an intracellular C-terminal region and seven transmembrane domains, linked by three extra- and three intracellular loops. The catecholamine ligand binding domain, studied using affinity-labeling and site-directed mutagenesis, is a pocket lined by residues belonging to the transmembrane domains. The region responsible for the interaction with the Gs protein which, when activated, stimulates adenylyl cyclase, is composed of residues belonging to the parts most proximal to the membrane of intracellular loop i3 and the C-terminal region. The pharmacology of the three subtypes is quite distinct: in fact most of the potent beta 1/beta 2 antagonists (the well known beta blockers) act as agonists on beta 3. The subtype is resistant to short-term desensitization mediated by phosphorylation through PKA or beta ARK, in stark contrast to the beta 1 or beta 2 subtypes. Various compounds (dexamethasone, butyrate, insulin) upregulate beta 1 or beta 2 subtypes while down-regulating beta 3 whose expression strictly correlates with differentiation of 3T3-F442A fibroblasts into adipocytes, thus confirming that the expression of the three subtypes may each be regulated independently to exert a specific physiologic role in different tissues or at different stages of development.

The functional effects of long-term activation of beta-adrenoceptors were investigated by measuring adenylyl cyclase activity, cyclic AMP accumulation and cyclic AMP-dependent protein kinase activity in CHW and L cells expressing either human beta 2- or beta 3-adrenoceptors. Pre-incubation of CHW and L cells expressing beta 2-adrenoceptors with 10 microM isoprenaline for 24 h produced a marked reduction in the total receptor number and dramatically reduced the capacity of the receptor to stimulate adenylyl cyclase maximally. In contrast, the ability of beta 3-adrenoceptors number was observed in L but not in CHW cells. Maximal levels of intracellular cyclic AMP concentrations were reached during the first hour of receptor activation with isoprenaline in all four cell lines. In the absence of phosphodiesterase inhibitors, cyclic AMP decreased to basal levels within 24 h of continuous stimulation. This phenomenon occurred more rapidly in cells expressing the beta 2- than the beta 3-adrenoceptors. These results confirm that, at the level of adenylyl cyclase stimulation and cyclic AMP accumulation, the beta 3-adrenoceptor is more resistant than the beta 2-adrenoceptor to long-term desensitization. However, when cyclic AMP-dependent protein kinase activity was considered, a 24 h stimulation of beta 2- and when cyclic AMP-dependent protein kinase activity was considered, a 24 h stimulation of beta 2- and beta 3-adrenoceptor expressing cells led to the desensitization of the kinase in L but not in CHW cells. In conclusion, long-term desensitization may have distinct functional effects on cell signalling depending on the receptor subtype and the cell type considered. These findings might have practical implications for future strategies involving long-term therapies with receptor agonists.

The role of brown adipose tissue (BAT) in human metabolism and its potential as an anti-obesity target organ have recently emerged. Following recent radiological detection of substantial amounts of BAT in adults by several independent research groups, an increasing number of studies are now dedicated to uncover BAT’s genetic, developmental and environmental determinants. In contrast to the murine BAT, human BAT is not present as a prominent depot in a well-defined location. The diffuse distribution of BAT in several areas in the body significantly limits its availability to research. Human brown adipose tissue cells lines are, therefore, critical in broadening the options available to researchers in the field. The human brown adipose tissue cell line PAZ6 was created to address such demand and has been well characterized by several research groups around the world. In the present review, we discuss their findings and propose potential applications of the PAZ6 cells in addressing the relevant questions in the brown adipose tissue field, namely for future use in therapeutic applications.

While the mitogen-activated protein kinase (MAPK) pathway coupled to receptor tyrosine kinases has been largely clarified, little is known about MAPK activation mediated by heterotrimeric G protein-coupled receptors. In a previous study, it has been shown that endothelin-1 (ET-1) signaling through heterotrimeric G protein-coupled receptors stimulates MAPK activity in primary cultures of astrocytes (Cazaubon, S., Parker, P. J., Strosberg, A.D., and Couraud, P.O. (1993) Biochem. J. 293, 381-386). To clarify the molecular mechanism responsible for this response, involvement of the adapter proteins, Shc and Grb2, has now been investigated. It is shown here that in these cells, ET-1 stimulates tyrosine phosphorylation of Shc, resulting in its stable association with Grb2 but not with Grb3-3, a Grb2 isoform with partially deleted SH2 domain. These results demonstrate that tyrosine-phosphorylated Shc specifically interacts with the SH2 domain of Grb2. This response was rapid and transient, showing a maximum at 10 min and declining at 60 min. Interestingly, direct activation of G proteins by fluoroaluminate mimics the ET-1 effect. In addition, a shift to a higher apparent molecular mass of Raf-1 kinase, likely reflecting its hyperphosphorylation, was also detected in ET-1-treated cells. These data strongly suggest that ET-1-induced MAPK activation is a G protein-coupled pathway that involves Shc, Grb2, and probably Raf-1. In conclusion, the Shc-Grb2 complex may be involved in the activation of the MAPK pathway, not only by several receptor tyrosine kinases but also by heterotrimeric G protein-coupled receptors, such as ET-1 receptors.

Myeloperoxidase is virucidal to human immunodeficiency virus type 1 (HIV-1) in the persistently infected CEM human T-cell line or in acutely infected human peripheral blood mononuclear cells, as judged by viral infectivity and P24 radioimmunoassay. HIV-1 was specifically inactivated by low doses of the human myeloperoxidase (1.4 to 14.3 mU/ml) and the cells were spared. A higher enzyme concentration (143 mU/m) was cytotoxic, but uninfected CEM cells and normal lymphocytes were resistant to > or = 143 mU of myeloperoxidase per ml. The enzyme was virucidal with the Cl- present in medium and did not require exogenous H2O2. Catalase, an antioxidant enzyme, partially inhibited the virucidal effect of myeloperoxidase. Hence, the H2O2 probably came from the HIV-infected cells themselves. These in vitro findings indicate that the myeloperoxidase system is capable of inactivating HIV-1 of infected cells.

Inflammatory diseases of the central nervous system, such as multiple sclerosis or experimental autoimmune encephalomyelitis, are characterized by adhesion of lymphocytes on cerebral microvascular endothelium, followed by transendothelial migration into the brain parenchyma. T lymphocyte adhesion to vascular endothelial cells is mediated by several types of adhesion molecules, including the integrin leukocyte function-associated molecule 1 and its endothelial counter receptor intercellular adhesion molecule 1 (ICAM-1), of the immunoglobulin superfamily. In order to understand the molecular mechanisms that support lymphocyte extravasation, we intended to investigate a putative role of ICAM-1 in signal transduction in brain microvessel endothelial cells. Here we describe, using a well differentiated rat brain endothelial cell line (RBE4 cells), that ICAM-1 activation by a specific monoclonal antibody, or by syngeneic encephalitogenic T cells, induces tyrosine phosphorylation of several proteins together with stimulation of the tyrosine kinase p60src activity. One of the major tyrosine-phosphorylated proteins, of 85 kDa, has been identified by immunoprecipitation and immunoblotting, as the recently described actin-binding protein, p60src substrate, cortactin. These findings demonstrate that ICAM-1 activation transduces signals in brain endothelial cells, which may lead to cytoskeleton changes and transendothelial migration of lymphocytes into the brain.

Rat brain microvessel endothelial cells were immortalized by transfection with a plasmid containing the E1A adenovirus gene. One clone, called RBE4, was further characterized. These cells display a nontransformed phenotype and express typical endothelial markers, Factor VIII-related antigen and Bandeiraea simplicifolia binding sites. When RBE4 cells were grown in the presence of bFGF and on collagen-coated dishes, confluent cultures developed sprouts that extend above the monolayer and organized into three-dimensional structures. The activity of the blood-brain barrier-associated enzyme, gamma-glutamyl transpeptidase (gamma GTP), was expressed in these structures, not in the surrounding monolayer. Similar results were obtained with the microvessel-related enzyme alkaline phosphatase (ALP). Addition of agents that elevate intracellular cAMP reduced the formation of three-dimensional structures, but every cell inside the aggregates still expressed gamma GTP and ALP activities. Such structures, associated with high levels of gamma GTP and ALP activities, were also induced by astroglial factors, including (1) plasma membranes from newborn rat primary astrocytes or rat glioma C6 cells, (2) C6 conditioned media, or (3) diffusible factors produced by primary astrocytes grown in the presence of, but not in contact with RBE4 cells. RBE4 cells thus remain sensitive to angiogenic and astroglial factors for the expression of the blood-brain barrier-related gamma GTP activity, as well as for ALP activity, and could constitute the basis of a valuable in vitro model of the blood-brain barrier.

3T3-F442A adipocytes, which express major beta 3-adrenergic receptors (beta 3-AR) (90%) and minor beta 1-AR (

The developmental pattern and laminar distribution of beta 1 and beta 2 adrenergic receptor subtypes were studied in cat visual cortex with autoradiography using [125I]iodocyanopindolol as a ligand and also with immunocytochemistry using a monoclonal antibody directed against beta adrenergic receptors. In the primary visual cortex of adult cats, the laminar distributions of both beta 1 and beta 2 adrenergic receptors revealed by autoradiography were very similar, with concentrations in layers I, II, III and VI. In young kittens (postnatal days 1 and 10), fewer beta adrenergic receptors were present, and they were concentrated in the deep cortical layers (V-VI) and subcortical white matter. Between postnatal days 15 and 40, beta adrenergic receptors increased in density more quickly in the superficial layers than they did in the deep and middle cortical layers. By postnatal day 40, the adult pattern was achieved, with two bands of intense binding in the superficial and deep cortical layers and a lower density in layer IV. Immunocytochemical techniques applied to adult cat cortex showed that beta adrenergic receptor-like immunoreactivity was found in different populations of neurons and glial cells. The immunoreactive neural cells were most dense in layers II, III and VI. About 50% of these immunoreactive neural cells were glial cells, primarily astrocytes. Immunoreactive pyramidal cells were mostly located in layers III and V. In layer IV, many stellate cells were stained. Immunoreactive astrocytes in the subplate and white matter progressively increased in number during development until adulthood. The pattern of laminar distribution and the developmental process was not affected by interrupting noradrenergic innervation from locus coeruleus either before or after the critical period. However, when visual input was interrupted by lesions of the lateral geniculate nucleus in young kittens (postnatal day 10), the density of both beta adrenergic receptor subtypes decreased significantly in the deep cortical layers. Lateral geniculate nucleus lesions in adult cats resulted in a pronounced decrease in beta adrenergic receptor density in layer IV.

The present investigation analyzes the cellular distribution of muscarinic acetylcholine receptors (mAChRs) and the gamma isoform of protein kinase C (PKC) in the rat parietal cortex employing the monoclonal antibodies M35 and 36G9, respectively. Muscarinic cholinoceptive neurons were most present in layers 2, 3 and 5, whereas most PKC gamma-positive cells were found in layers 2, 5 and 6. Under normal, non-stimulated conditions, approximately 58% of all muscarinic cholinoceptive neurons were immunoreactive for PKC gamma. Conversely, nearly all PKC gamma-positive neurons were M35-immunoreactive. Although both pyramidal and nonpyramidal neurons express the two types of protein, the pyramidal cell type represents the vast majority. Of all cortical neurons, the large (15-25 microns in diameter) muscarinic cholinoceptive pyramidal neurons in layer 5 express the gamma isoform of PKC most abundantly and most frequently. Approximately 96% of these cells are immunoreactive for PKC gamma. Stimulation of mAChRs by the cholinergic agonist carbachol resulted in a pronounced increase in the intensity of 36G9 immunoreactivity, which may suggest that the mAChRs are functionally linked to the colocalized PKC gamma. No change was found in the number of 36G9-immunoreactive neurons. In contrast, the number of immunocytochemically detectable muscarinic cholinoceptive neurons increased by approximately 38% after carbachol stimulation. The high degree of codistribution in cortical neurons of both transduction proteins suggests a considerable cholinergic impact upon the regulation of PKC gamma, a candidate key enzyme in cortical learning and memory mechanisms.

Endothelin (ET)-1 was originally characterized as a potent vasoconstrictor peptide secreted by vascular endothelial cells. It possesses a wide range of biological activities within the cardiovascular system and in other organs, including the brain. Also secreted by endothelial cells, nitric oxide (NO), has recently been identified as a relaxing factor, as well as a pleiotropic mediator, second messenger, immune defence molecule, and neurotransmitter. Most of the data concerning the secretion of these two agents in vitro has been collected from studies on macrovascular endothelial cells. Given the remarkable heterogeneity of endothelia in terms of morphology and function, we have analyzed the ability of brain microvessel endothelial cells in vitro to release ET-1 and NO, which, at the level of the blood-brain barrier, have perivascular astrocytes as potential targets. The present study was performed with immortalized rat brain microvessel endothelial cells, which display in culture a non transformed phenotype. Our data demonstrate that: (1) these cells release NO when induced by IFN gamma and TNF alpha, (2) they constitutively secrete ET-1, and (3) cAMP potentiates the cytokine-induced NO release and exerts a biphasic regulation on ET-1 secretion: micromolar concentrations of 8-Br-cAMP inhibit and higher doses stimulate ET-1 secretion. This stimulation is blocked by EGTA and the calmodulin antagonist W7, but not by protein kinase C inhibitors, suggesting the involvement of the calmodulin branch of the calcium messenger system. These results suggest that cerebral microvessel endothelial cells may participate in vivo to the regulation of glial activity in the brain through the release of NO and ET-1.

Endothelin receptors have been identified on astrocytes and astrocytoma, but their physiological significance has remained elusive. It is shown here that endothelins induce c-fos in primary cultures of mouse embryo astrocytes, as well as in two subclones of rat astrocytoma C6 cells, although with different kinetics. In addition, nerve growth factor expression is stimulated, as seen by mRNA accumulation and protein secretion, in primary astrocytes and one of the two C6 subclones, with an apparent correlation with the transience of c-fos induction. The activation of protein kinase C appears as an obligatory step during these processes, because (a) inhibition of protein kinase C by staurosporine blocks the induction by endothelin or phorbol esters of both c-fos and nerve growth factor, and (b) phorbol ester-evoked down-regulation of protein kinase C completely abolishes the c-fos induction by endothelin, but not that by the beta-adrenergic agonist isoproterenol, a known activator of the cyclic AMP-dependent pathway. Our results support the hypothesis that c-fos product might be implicated in nerve growth factor expression by astrocytes, and also suggest that endothelins may participate in vivo in the modulation of the glial neurotrophic activity during brain development or wound healing.

A novel screening assay for the identification of baculovirus infected cells expressing membrane receptors was developed by using a replica transfer technique. Sf9 cells were cotransfected with wild type baculoviral DNA and the transfer vector pVL941-beta 1 containing the coding region of the human beta 1-adrenergic receptor gene. Infected cells embedded in agarose were incubated with [125I]-iodocyanopindolol and transferred onto filters that were subsequently autoradiographed. This procedure resulted in the isolation of recombinant baculoviruses that expressed beta 1-adrenergic receptors. Binding assays carried out with [125I]-ICYP indicated that more than 600,000 receptors were expressed per cell, the highest level noted so far for this receptor in genetically engineered cells. Sf9 cells expressing the beta 1-AR were analysed by ligand binding, competition experiments, adenylyl cyclase stimulation and photoaffinity labeling. These cells express a homogenous population of receptors and display the known pharmacological properties of beta 1-AR in human tissues.

Glucose oxidase and peroxidase (lactoperoxidase or myeloperoxidase) are virucidal to human immunodeficiency virus type 1 (HIV-1) in the presence of sodium iodide, as assessed by the loss of viral replication in a syncytium-forming assay or by the inhibition of cytopathic effects on infected cells. In the presence of low concentrations of sodium iodide, five HIV-1 isolates were equally susceptible to this virucidal system at enzyme concentrations of a few milliunits. The loss of viral replication was linearly related to the time of incubation in the enzyme solutions, with an inactivation rate of 1 log unit every 30 min. These enzymes and this halide were also cytotoxic to chronically infected, but not to uninfected, cultured CEM cells. Protein conjugates were prepared by using the enzymes and murine antibody 105.34, which recognized the V3 loop of HIV-1 LAI isolate surface glycoprotein, or recombinant human CD4. The protein conjugates inactivated free virus at rates similar to those of the free enzymes and were more effective than antibody or recombinant CD4 alone. These in vitro findings demonstrate that the peroxidase-H2O2-halide system provides potent virucidal activity against HIV-1.

SUMMARY It has been reported that autoantibodies against the β2-adrenergic receptors are involved in the pathology of allergic disorders and of Chagas' disease. Therefore, the immune response against a peptide (H26Q) corresponding to the putative second extracellular loop of the human β2-adrenergic receptor, which could be a target for autoantibody attack, was analysed in view of its possible immunogenicity. The free peptide induced a T cell-mediated humoral response in the context of three different murine MHC haplotypes. The T cell epitope was found to be localized in the N-terminal region of the peptide. Highly specific T helper cells were capable of stimulating B cells with the potential to generate a large antibody repertoire reactive with the loop peptide. MoAbs were screened to analyse this B cell response for antibodies potentially interfering with receptor function and a MoAb was found that impaired ligand binding to the receptor.

In duck erythroblasts, two major populations of untranslated messenger (m) RNP can be separated by sucrose gradient centrifugation in low ionic strength. One of these contains globin mRNA associated to protein factors, among them the prosomes. The other, sedimenting in the 35S zone, contains non-globin mRNA. From this '35S' mRNP, a new RNP particle called the prosome-like particle was isolated and characterized [Akhayat, O., Infante, A. A., Infante, D., Martins de Sa, C., Grossi de Sa, M.-F.Scherrer, K. (1987) Eur. J. Biochem. 170, 23-33]. The PLP is a multimer of a protein of M(r) 21,000, and contains small RNA species. The particle is tightly associated with repressed mRNA and inhibits in vitro protein synthesis. We show here that the protein of M(r) 21,000, constituting the prosome-like particle, is apoferritin. Different approaches confirm the RNP character of this particle and provide evidence that some of its RNA species are tRNA. The hypothesis is discussed as to whether (apo-)ferritin might serve other functions in addition to iron storage.

SM-11044 (L-threo-3-(3,4-dihydroxyphenyl)-N-[3-(4-fluorophenyl) propyl] serine pyrrolidine amide hydrobromide) stimulated the relaxation of guinea pig ileum (EC50: 3.0 × 10−8 M), trachea (EC50: 1.3 × 10−7 M), lung parenchyma (EC50: 2.1 × 10−6 M) and the increase in right atrial rate (EC50: 6.9 × 10−6 M). It also induced lipolysis of rat white adipocytes (EC50: 1.2 × 10−6 M). Both the relaxant response of ileum and the lipolytic response of adipocytes to SM-11044 were resistant to inhibition by propranolol (10−6 M), but were antagonized by cyanopindolol (10−6 M). In contrast, the responses to SM-11044 in trachea, lung parenchyma and right atrium were almost completely abolished by propranolol (10−6 M). Furthermore, the selectivity of SM-11044 relative to isoproterenol was ileum > adipocytes >trachea (β2) > lung (β2) > atrium (β1). These results suggest that SM-11044 is a selective agonist of atypical β-adrenoceptors that are resistant to antagonism by propranolol but sensitive to cyanopindolol. The receptor binding and adenylate cyclase stimulating activity of SM-11044 were also examined on transfected Chinese hamster ovary cells expressing human β1-, β2- or β3-adrenoceptors. SM-11044 inhibited the binding of [125I]iodocyanopindolol to the three types of receptors in a concentration-dependent manner. The selectivity in terms of Ki values was β3(Ki: 1.3 × 10−6 M) >β2 (Ki: 4.1 × 10−6 M) >β1 (Ki: 18.1 × 10−6 M)-adrenoceptors. SM-11044 stimulated cAMP accummulation in these three types of cells in a concentration-dependent manner. These results indicate that SM-11044 is a new atypical β-adrenoceptor agonist in the human, the guinea pig and rat.

Signaling through serotonin 5-HT1A receptors involves multiple pathways. We have investigated the functional coupling of the human 5-HT1A receptor to different G proteins using an in vitro reconstitution system based on the expression of recombinant receptor (r5-HT1A) and G alpha-subunits (rG alpha) in Escherichia coli. The r5-HT1A receptor was expressed by insertion in a vector allowing its active expression in E. coli inner membranes. Binding of the selective agonist [3H] +/- 8-hydroxy-(2-N-dipropylamine)tetralin ([3H]8-OH-DPAT) to intact bacteria or E. coli membranes was saturable with a KD of approximately 8 nM and an average of 120 sites/bacterium. Binding properties of several serotoninergic ligands to r5-HT1A receptors were comparable with those measured in mammalian cells. Incubation of rG alpha.beta gamma with E. coli membranes resulted in high affinity agonist [3H]8-OH-DPAT binding (KD = 0.7 nM) and titration with a panel of rG alpha subtypes showed the order of potency: rGi alpha-3 greater than rGi alpha-2 greater than rGi alpha-1 much greater than rGo alpha, while rGs alpha appeared incapable of interacting with 5-HT1A receptors. Moreover, agonist-mediated enhancement of [35S]guanosine 5'-O-(3-thiotriphosphate) binding to rGi alpha confirmed the achievement of the functional interaction between receptor and G proteins. Our findings are in agreement with the in vivo ability of 5-HT1A receptors to activate Gi alpha related to adenylyl cyclase inhibition or K+ channel activation, but do not support previously reported adenylyl cyclase stimulation through interaction with Gs alpha.

Hepatitis C virus (HCV) nucleocapsid assembly requires dimerization of the core protein, an essential step in the formation of the virus particle. We developed a novel quantitative assay for monitoring this protein–protein interaction, with the goal of identifying inhibitors of core dimerization that might block HCV production in infected Huh-7.5 hepatoma cells. Two core-derived, 18-residue peptides were found that inhibited the dimerization of a fragment of core comprising residues 1–106 (core106) by 68 and 63 %, respectively. A third, related 15-residue peptide displayed 50 % inhibition, with an IC50 of 21.9 μM. This peptide was shown, by fluorescence polarization, to bind directly to core106 with a K d of 1.9 μM and was displaced by the unlabelled peptide with an IC50 of 18.7 μM. When measured by surface plasmon resonance, the same peptide bound core169 with a K d of 7.2 μM. When added to HCV-infected cells, each of the three peptides blocked release, but not replication, of infectious virus. When measured by real-time RT-PCR, the RNA levels were reduced by 7-fold. The 15-residue peptide had no effect on HIV propagation. Such inhibitors may constitute useful tools to investigate the role of core dimerization in the virus cycle.

Expression of ligand binding properties for an atypical beta-adrenergic receptor (beta-AR) subtype was studied during the adipose differentiation of murine 3T3-F442A cells and compared with that of the human beta 3-AR expressed in Chinese hamster ovary cells stably transfected with the human beta 3-AR gene (CHO-beta 3 cells) Emorine, L. J., Marullo, S., Briend-Sutren, M. M., Patey, G., Tate, K., Delavier-Klutchko, C., and Strosberg, A. D. (1989) Science 245, 1118-1121). 3T3-F442A adipocytes exhibited high and low affinity binding sites for (-)-4-(3-t-butylamino-2-hydroxypropoxy) [5,7-3H]benzimidazole-2-one ((-)-[3H]CGP-12177) (KD = 1.2 and 38.3 nM) and (-)-[125I]iodocyanopindolol ([125I]CYP) (KD = 47 and 1,510 pM). The high affinity sites corresponded to the classical beta 1- and beta 2-AR subtypes whereas the KD values of the low affinity sites for the radioligands were similar to those measured in CHO-beta 3 cells (KD = 28 nM and 1,890 pM for (-)-[3H]CGP12177 and [125I]CYP, respectively). These low affinity sites were undetectable in preadipocytes but represented about 90% of total beta-ARs in adipocytes. The atypical beta-AR and the human beta 3-AR add similarly low affinities (Ki = 3-5 microM) for (+/-)-(2-(3-carbamoyl-4-hydroxyphenoxy)ethylamino-3)-(4-(1-methyl- 4- trifluormethyl-2-imidazolyl)-phenoxy)-2-propanol methane sulfonate (CGP20712A) or erythro-(+/-)-1-(7-methylindan-4-yloxy)-3-isopropylaminob utan-2-ol (ICI118551), highly selective beta 1- and beta 2-AR antagonists, respectively, in agreement with the poor inhibitory effect of the compounds on (-)-isoproterenol (IPR)-stimulated adenylate cyclase activity. Atypical beta-AR and beta 3-AR had an affinity about 10-50 times higher for sodium-4-(2-[2-hydroxy-2-(3-chlorophenyl)ethylamino]propyl)phenoxyace tate sesquihydrate (BRL37344) than the beta 1-AR subtype. This correlates with the potent lipolytic effect of BRL37344 in adipocytes. The rank order of potency of agonists in functional and binding studies was BRL37344 greater than IPR less than (-)-norepinephrine greater than (-)-epinephrine both in 3T3 adipocytes and CHO-beta 3 cells. As in CHO-beta 3 cells, the classical beta 1- and beta 2-antagonists CGP12177, oxprenolol, and pindolol were partial agonists in adipocytes. Although undetectable in preadipocytes, a major mRNA species of 2.3 kilobases (kb) and a minor one of 2.8 kb were observed in adipocytes by hybridization to a human beta 3-AR specific probe.(ABSTRACT TRUNCATED AT 400 WORDS)

Early passage bovine brain capillary endothelial cells were immortalized by transfection with the plasmid pSV3 neo. Cells from one clone, SV-BEC, expressed nuclear SV 40 large T antigen, displayed a contact-inhibited and anchorage-dependent proliferation, and a high sensitivity to the addition of exogenous basic fibroblast growth factor. SV-BEC cells are morphologically unaltered and express typical markers of endothelial cells: Factor VIII-related antigen, angiotensin-converting enzyme and Griffonia simplicifolia agglutinin binding site. Endothelium like immunoreactivity was detected in the conditioned medium from these cells. Moreover, SV-BECs present numerous intercellular tight junctions characteristic of the blood-brain barrier and possess functional beta 1- and beta 2-adrenergic receptors, as observed on isolated bovine brain capillaries.

The density of endothelin-1 (ET-1) receptors on rat astrocytoma C6 cells is down-regulated by activation of protein kinase C (PKC) [6]. We have investigated whether intracellular accumulation of cyclic adenosine monophosphate (cAMP) may also modulate surface ET-1 receptor number. The density of ET-1 receptors was measured by binding of [ 125 I]ET-1 on rat astrocytoma C6 intact cells exposed to catecholamines, dibutyryl-cAMP or forskolin. Prolonged exposure of the cells to the β-adrenergic agonists, isoproterenol or noradrenaline, results in a time- and dose-dependent decrease in cell surface ET-1 receptor number. This decrease proceeds slowly: maximal down-regulation is obtained by 6–7 h and sustained for up to 24 h in the presence of 10 μM isoproterenol. Since this down-regulation is mimicked by dibutyryl-cAMP (4 μM) and by forskolin (10 μM), we conclude that ET-1 receptors are susceptible to down-regulation through a cAMP-dependent pathway.

This study describes the colocalization of muscarinic acetylcholine receptors (mAChRs) and the neuropeptide somatostatin (SOM) in nonpyramidal neurons of the rat dorsal hippocampus. SOM and mAChRs were identified by immunocytochemistry employing antibody S309 and M35, respectively. Half of the SOMergic cell population is found to be immunoreactive for muscarinic receptor protein as obtained by fluorescent double-labeling techniques. These findings provide additional evidence for a direct cholinergic influence upon SOMergic, nonpyramidal neurons, and defines the anatomical distribution of SOMergic, cholinoceptive neurons in the dorsal hippocampus. Concerning the muscarinic cholinoceptive, nonpyramidal neuron population of the dorsal hippocampus, a considerable number (approximately one-third) was found to be colocalized with somatostatin. These results indicate that a significant part of the cholinergic influence upon hippocampal nonpyramidal neurons is relayed via SOMergic neurons.

In the present study muscarinic and nicotinic cholinergic receptors in the SCN region were demonstrated and analyzed, employing monoclonal antibodies to purified muscarinic and nicotinic cholinergic receptor proteins. A near-total colocalization of the two acetylcholine receptor subclasses in cholinoceptive neurons of the SCN area was found. The antibodies applied to aging rat brain (at 30-34 months) revealed a clear decrease in immunoreactivity in senescence albeit with a high level of individual variability. Furthermore, in 8 out of 10 aged animals examined a considerable increase of astrocytes possessing muscarinic cholinergic receptors was observed.

The structural properties of a number of proteins belonging to the family of receptors coupled to GTP binding proteins are discussed in relation to their function. The structure of the ligand binding site and of the regions involved in coupling to the G proteins are analyzed mainly for the adrenergic and muscarinic cholinergic receptors, for which site-directed mutagenesis and chimaeric constructions have been studied. The structure of the genes are compared and the presence of various regulatory elements is discussed in relation to control of expression. Mechanism of desensitization and internalization, while mostly studied for the beta 2-adrenergic receptor, are proposed to be generally applicable to all G-protein-coupled receptors.

We present a homology scanning microcomputer program to predict functional T-cell epitopes within proteins. By taking into account particular human or mouse restriction elements the predictions are made haplotype-specific. The generality of this approach is confirmed by (i) identification of well-characterized immunogenic T-cell determinants in lysozyme (ii) search for potential T epitopes on unanalysed proteins like the human beta 2-adrenoreceptor (iii) modification of non-immunogenic peptide sequences in order to generate T-cell determinants.

The presence and properties of serum autoantibodies against beta-adrenergic receptors in patients with idiopathic dilated cardiomyopathy were studied using synthetic peptides derived from the predicted sequences of the human beta-adrenergic receptors. Peptides corresponding to the sequences of the second extracellular loop of the human beta 1- and beta 2-adrenergic receptors were used as antigens in an enzyme immunoassay to screen sera from patients with dilated cardiomyopathy (n = 42), ischemic heart disease (n = 17), or healthy blood donors (n = 34). The sera of thirteen dilated cardiomyopathy patients, none of the ischemic heart disease patients, and four of the healthy controls monospecifically recognized the beta 1-peptide. Only affinity-purified antibodies of these patients had a inhibitory effect on radioligand binding to the beta 1 receptor of C6 rat glioma cells. They recognized the receptor protein by immunoblot and bound in situ to human myocardial tissue. We conclude that a subgroup of patients with idiopathic dilated cardiomyopathy have in their sera autoantibodies specifically directed against the second extracellular loop of the beta 1-adrenergic receptor. These antibodies could serve as a marker of an autoimmune response with physiological and/or pathological implications.

Monoclonal antibody II-6-18 recognizes a serogroup-1-specific Legionella pneumophila antigenic determinant which has been shown to be virulence-associated. We previously reported the physicochemical characterization by means of a quantitative fluorometric assay of monoclonal antibody II-6-18 binding to L. pneumophila, and its implications concerning the nature of the antigen. We describe here the isolation and the purification of the antigen by chemical and immunological methods, followed by its partial chemical analysis. The results demonstrate that the epitope - an immunodominant carbohydrate which includes a fucosamine-like residue — is part of the cell wall lipopolysaccharide (LPS). It is localized in the polysaccharide moiety of the LPS which contains KDO, rhamnose, mannose, glucosamine and an unidentified aminodideoxyhexose X1, but no heptose. The aminodideoxyhexose X1 could be fucosamine and is probably the immunodominant residue in the epitope, localized, at least partially, at the end of the polysaccharide chain.