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A D Strosberg

Publications and source records attributed to A D Strosberg.

At least 19 recordsLinked to original sources
PubMed1992

Inhibition by dexamethasone of beta 3-adrenergic receptor responsiveness in 3T3-F442A adipocytes. Evidence for a transcriptional mechanism.

Modulation of beta 3-adrenergic receptor (beta 3AR) expression by dexamethasone was investigated in the murine 3T3-F442A adipocytic cell line. In untreated cells, a major population of binding sites (62,000-114,000 sites/cell) of low affinity for (-)-[3H] CGP12177 and (-)-[125I]iodocyanopindolol (corresponding to the beta 3AR subtype) was present along with a minor population (6,500-8,000 sites/cell) of sites of high affinity for the radioligands (corresponding to a mixture of the beta 1 and beta 2AR subtypes). Long-term exposure of the cells to 250 nM dexamethasone led to a sharp decrease in beta 3AR density (less than 5,000 sites/cell) which paralleled a diminished potency of the beta 3AR-selective agonists BRL37344 and CGP12177 to stimulate the production of intracellular cAMP. Analysis of RNA by polymerase chain reaction and nuclear run-on assays indicated that dexamethasone inhibited the synthesis of beta 3AR mRNA, resulting in 4-8-fold decrease in the steady-state levels of this mRNA. The down-regulation of beta 3AR protein and cellular mRNA appeared to be mediated by the receptor for glucocorticoids as assessed by the antagonistic action of the anti-glucocorticoid RU38486.

3T3 Cells↗
PubMed1992

The protein of M(r) 21,000 constituting the prosome-like particle of duck erythroblasts is homologous to apoferritin.

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.

Amino Acid Sequence↗
PubMed1992

Molecular characterization of a human anti-Rh(D) antibody with a DH segment encoded by a germ-line sequence.

The lambda-light-chain and lambda-heavy-chain variable-region genes of an anti-Rh(D) (Rh, Rhesus; D, heavy-chain diversity region) human monoclonal antibody secreted by lymphocytes transformed by the Epstein-Barr virus have been cloned and sequenced. Sequence comparison of the anti-Rh(D)mAb lambda-chain variable region with those of the other available human lambda chains revealed that it belonged to the human V lambda I (V lambda, variable region of lambda chain) subgroup. The greatest sequence similarity (80%) was observed with that of another anti-Rh antibody lambda-chain directed against the Rh(c) antigen. For the VH (VH, variable region of heavy chain) sequence, the highest similarity (86%) was observed with the germline VHG3 gene which belongs to the VHI subgroup. The expressed DH sequence of the anti-Rh(D) antibody is also of germline origin and complementarity-determining region 3 is thus produced by VH-DH and DH-JH (J, joining region) joining without recombination of multiple DH gene segments.

Amino Acid Sequence↗
PubMed1992

In vitro study of a novel atypical beta-adrenoceptor agonist, SM-11044.

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 x 10(-8) M), trachea (EC50: 1.3 x 10(-7) M), lung parenchyma (EC50: 2.1 x 10(-6) M) and the increase in right atrial rate (EC50: 6.9 x 10(-6) M). It also induced lipolysis of rat white adipocytes (EC50: 1.2 x 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 greater than adipocytes greater than trachea (beta 2) greater than lung (beta 2) greater than atrium (beta 1). These results suggest that SM-11044 is a selective agonist of atypical beta-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 beta 1-, beta 2- or beta 3-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 beta 3 (Ki: 1.3 x 10(-6) M) greater than beta 2 (Ki: 4.1 x 10(-6) M) greater than beta 1 (Ki: 18.1 x 10(-6) M)-adrenoceptors.(ABSTRACT TRUNCATED AT 250 WORDS)

Adenylyl Cyclases↗
PubMed1992

Functional expression of the human serotonin 5-HT1A receptor in Escherichia coli. Ligand binding properties and interaction with recombinant G protein alpha-subunits.

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.

8-Hydroxy-2-(di-n-propylamino)tetralin↗
PubMed1992

Morphology and distribution of neurons and glial cells expressing beta-adrenergic receptors in developing kitten visual cortex.

The morphology and distribution of cells expressing beta-adrenergic receptors has been studied in developing kitten visual cortex using a monoclonal antibody which recognizes both beta-1 and beta-2 adrenergic receptors. We found specific populations of neurons and glial cells which express beta-adrenergic receptor immunoreactivity in the kitten visual cortex. In adult animals, the receptors are most concentrated in the superficial and deep cortical layers (layers I, II, III and VI). About 50% of the stained neural cells in adult cat visual cortex are glial cells. Most of the immunoreactive neurons in layers III and V are pyramidal cells while those in layers II and IV are more likely to be nonpyramidal cells. In neonatal kittens, staining is weaker than that in adult cats and it appears to be concentrated in neurons of the deep cortical layers and in the subcortical plate and white matter. Only a few immunoreactive glial cells were found at this age. Receptor numbers increase after birth and by 24 days of age, the laminar distribution of beta-adrenergic receptors approaches that of adult animals. Immunoreactive glial cells in the white matter show a progressive increase in number throughout postnatal development.

Aging↗
PubMed1992

Immunocytochemical localization of muscarinic acetylcholine receptors in the rat endocrine pancreas.

Immunocytochemical application of the antimuscarinic acetylcholine receptor antibody M35 to pancreas tissue revealed the target areas for the parasympathetic nervous system. Immunoreactivity in the endocrine pancreas was much higher than that in the exocrine part. Moreover, the endocrine cells at the periphery of the islets of Langerhans displayed the highest level of immunoreactivity. Based on these findings in the mantle of the islets, two types of islets have been distinguished: type-I islets with intensely stained mantle cells, and type-II islets with a much lower concentration of these cells. On average, type-I islets were larger (244.8 microns +/- 6.1 SEM) than type-II islets (121.5 microns +/- 3.8 SEM). M35-immunoreactivity was present on the majority of D cells, which were characterized by their immunoreactivity to somatostatin [of 446 D cells 356 (79.8%) were M35-immunopositive]. However, only a small proportion of the intensely stained mantle cells belonged to the D cell population. Therefore, it is concluded that the majority of the intensely stained mantle cells represent glucagon-secreting A and/or pancreatic polypeptide-secreting F cells. The intensity of M35-immunoreactivity at the periphery and central core of the islets paralleled the density of cholinergic innervation, suggesting a positive correlation between the intensity of cholinergic transmission and the number of muscarinic acetylcholine receptors at the target structures. The present study further revealed some striking parallels for the muscarinic acetylcholine receptor characteristics between the (endocrine) pancreas and the central nervous system.

Animals↗
PubMed1992

Localisation of three epitopes of the env protein of feline immunodeficiency virus.

The envelope protein of the feline immunodeficiency virus (FIV) was analyzed using several epitope prediction programs based on profiles of hydrophilicity, antigenicity, and probability of residues to lie on the protein surface. Tentative homologies with the immunodominant epitope sites in simian virus (SIV) or human immunodeficiency virus (HIV) such as the V3 loop, the site of cleavage between surface envelope protein (SU) and transmembrane envelope protein (TM), and sites of N-glycosylation were thus identified. Five peptides corresponding to potential epitopes were synthesized. Four out of five peptides (P99, P100, P101, P103) were from the FIV surface envelope protein (SU). The last one (P102) was from the FIV transmembrane envelope protein TM. Three of these peptides (P99, P100, and P102) were recognized in ELISA by almost all the sera from infected cats. The peptide from TM (102) was recognized by sera from both naturally infected and inoculated cats, whereas peptides P99 and P100 (from SU) were recognized mainly by sera from naturally infected cats. On the basis of these results we propose that peptides P99, and P100 from SU and P102 from TM constitute epitopes on the FIV env protein.

Amino Acid Sequence↗
PubMed1992

Visualization of cholinoceptive neurons in the rat neocortex: colocalization of muscarinic and nicotinic acetylcholine receptors.

The present investigation analyzes the cellular distribution of muscarinic and nicotinic acetylcholine receptors in rat neocortex, by use of monoclonal antibodies raised against purified receptor proteins. The degree of colocalization of both types of receptors was determined by way of immunofluorescent double-labeling techniques. For both classes of receptors, pyramidal and nonpyramidal cells were found immunostained and an identical laminar distribution pattern of immunopositive neurons in the rat neocortex became apparent. A striking similarity in distribution of the two cholinergic receptor types was found in the frontal/motor and parietal cortex. Accordingly, we observed a high degree of colocalization of muscarinic and nicotinic acetylcholine receptors within immunopositive cortical neurons. Approximately 90% of the cholinoceptive neurons expressed both types of receptors. The current data demonstrate that (i) the distribution of muscarinic and nicotinic cholinoceptive neurons in the neocortex is present in identical laminar patterns and represent the same type of cells, (ii) both classes of cholinergic receptors are highly colocalized within cholinoceptive neurons, which points at individual neurons as a likely site of interaction between muscarinic and nicotinic acetylcholine receptor-mediated processes.

Animals↗
PubMed1992

A monoclonal antibody specific for conidia and mycelium wall layer of Penicillium and Aspergillus.

A monoclonal antibody was obtained from BALB/c mice immunized with Penicillium frequentans mycelium. The specificity of the antibody was evaluated by enzyme-linked immunosorbent and indirect immunofluorescence assays against the same mycelium. This IgM antibody cross-reacted with various strains of the Penicillium and Aspergillus genera. By indirect immunofluorescence assays, the antibody was able to stain about 10% of Penicillium and Aspergillus conidia, but major part of conidia did not absorb the fluorescence-labeled antibody before swelling. During germination of P. frequentans conidia, the germ tube wall which constitutes a continuation of an inner wall layer was also stained. During germination of P. griseofulvum, the protrusion of the germ tube wall was not always recognized by the antibody because the germ tube wall was constituted by a continuation of an outer spore wall layer. The study of the staining patterns of the spores and the protrusions suggests that the antibody specifically recognizes an antigen of the inner spore wall layer. The monoclonal antibody reacts with extracellular galactomannans produced by genera Aspergillus and Penicillium but is not directed against beta-(1,5)-linked galactofuranose units.

Animals↗
PubMed1992

Induction of a pharmacologically active clonotypic B cell response directed to an immunogenic region of the human beta 2-adrenergic receptor.

It has been reported that autoantibodies against the beta 2-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 beta 2-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.

Animals↗
PubMed1991

Atypical beta-adrenergic receptor in 3T3-F442A adipocytes. Pharmacological and molecular relationship with the human beta 3-adrenergic receptor.

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)

3T3 Cells↗
PubMed1991

Catecholamines stimulate the IFN-gamma-induced class II MHC expression on bovine brain capillary endothelial cells.

The brain has been considered for a long time as an immunologically privileged site because of the lack of a true lymphatic system and the existence of several barriers that isolate it from the periphery. In the last few years, it became evident that cells in the central nervous system (astrocytes, microglial cells, and brain capillary endothelial cells) can be induced to express class II MHC and present Ag to T lymphocytes. The brain capillary endothelial cells, which are strategically located at the interface between blood and brain, could be involved in the initiation of immune responses within the brain parenchyma. We have previously characterized bovine brain capillary endothelial cells in culture and shown that they maintain in vitro a fully differentiated phenotype associated with the blood-brain barrier endothelium. In order to assess the role of these cells in the development of immune responses in the brain, we initiated the present study on the regulation of their class II MHC surface expression. Our data indicate that this expression on bovine brain capillary endothelial cells is inducible by IFN-gamma and further stimulated by catecholamines through activation of beta-adrenergic receptors. However, this latter effect is not mimicked by forskolin, theophylline, or dibutyryl-cAMP, suggesting the involvement of a cAMP-independent mechanism.

3-Pyridinecarboxylic acid, 1,4-dihydro-2,6-dimethy↗
PubMed1991

cAMP-dependent down-regulation of endothelin-1 receptors on rat astrocytoma C6 cells.

The density of endothelin-1 (ET-1) receptors on rat astrocytoma C6 cells is down-regulated by activation of protein kinase C (PKC). 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 [125I]ET-1 on rat astrocytoma C6 intact cells exposed to catecholamines, dibutyryl-cAMP or forskolin. Prolonged exposure of the cells to the beta-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 microM isoproterenol. Since this down-regulation is mimicked by dibutyryl-cAMP (4 microM) and by forskolin (10 microM), we conclude that ET-1 receptors are susceptible to down-regulation through a cAMP-dependent pathway.

Animals↗
PubMed1991

Expression of two human beta-adrenergic receptors in Escherichia coli: functional interaction with two forms of the stimulatory G protein.

When expressed in Escherichia coli, the human beta 1- and beta 2-adrenergic receptors retain their ligand binding specificity. Their functional integrity was investigated by analyzing receptor-guanine nucleotide-binding regulatory (G) protein coupling by using two splice variants of the alpha subunit of the stimulatory G protein Gs synthesized in E. coli (rGs alpha-S and rGs alpha-L) and the beta gamma subunits of G protein purified from bovine brain. In competition binding experiments with (-)-[125I]iodocyanopindolol and (-)-isoproterenol, rGs alpha-S.beta gamma and rGs alpha-L.beta gamma reconstituted guanine nucleotide-sensitive high-affinity agonist binding with comparable affinities, whereas rGs alpha PT, a mutant of rGs alpha-L with an altered carboxyl terminus, and a recombinant subtype of the alpha subunit of the inhibitory G protein, rGi alpha-1, were approximately 20- and approximately 200-fold less potent, respectively. A comparison of the beta 1- and beta 2-adrenergic receptor expressed in E. coli with the beta 2-receptor in S49 murine lymphoma cyc- cell membranes revealed a similar affinity of rGs alpha-S and rGs alpha-L for the recombinant and native receptors. After stable incorporation of rGs alpha-S.beta gamma into E. coli membranes, receptor-G protein coupling was also verified by determining the isoproterenol-mediated acceleration of the rate for guanine 5'-[gamma-[35S]thio]triphosphate binding. These results show that (i) receptor-G protein coupling can be reconstituted in E. coli using recombinant components and that (ii) such an approach may be more generally used to evaluate coupling preferences between defined molecular species of receptors and G-protein subunits.

Cell Membrane↗
PubMed1991

Functional endothelin-1 receptors in rat astrocytoma C6.

Rat astrocytoma C6 cells have been recently identified as target cells for ET-1, which stimulates inositol lipid turnover in these cells. It is shown here that binding of ET-1 to high-affinity receptors on C6 cells leads to 40-45% inhibition of isoproterenol-induced intracellular cyclic AMP accumulation, as well as to stimulation of inositol lipid turnover, both effects characterized by an absolute requirement of extracellular calcium. Moreover, ET-1, which has been generally reported to have a mitogenic effect on a variety of target cells including primary rat astrocytes, is shown here to stimulate or, alternatively, inhibit DNA synthesis in C6 cells, depending on the subclone considered.

Animals↗
PubMed1991

Expression of three human beta-adrenergic-receptor subtypes in transfected Chinese hamster ovary cells.

The genes coding for three pharmacologically distinct subtypes of human beta-adrenergic receptors (beta 1 AR, beta 2 AR and beta 3 AR) were transfected for expression in Chinese hamster ovary (CHO) cells. Stable cell lines expressing each receptor were analyzed by ligand binding, adenylate cyclase activation and photoaffinity labeling, and compared to beta AR subtypes observed in previously described tissues, primary cultures and transfected cell lines. Each of the three receptor subtypes displayed saturable [125I]iodocyanopindolol-binding activity. They showed the characteristic rank order of potencies for five agonists, determined by measuring the accumulation of intracellular cAMP. These recombinant cell lines express a homogeneous population of receptors and display the known pharmacological properties of beta 1 AR and beta 2 AR, in human tissues. It is therefore likely that the pattern of ligand binding and adenylate cyclase activation, mediated by the new beta 3 AR in CHO cells, also reflects the yet-undetermined pharmacological profile in humans.

Adenylyl Cyclases↗