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At least 1,513 records · Page 84Linked to original sources
PubMed1990

Structure-activity relationships of monomeric and dimeric synthetic ACTH fragments in perifused frog adrenal slices.

The effect of synthetic monomeric and dimeric ACTH fragments on spontaneous and ACTH(1-39)-evoked steroidogenesis in frog interrenal tissue was studied in vitro. Infusion of ACTH fragment 11-24 (10(-6) M) or its dimeric conjugates, attached either by their N-terminal, Glu(11-24)2, or their C-terminal amino acid, (11-24)2Lys, had no effect on the spontaneous release of corticosteroids. The monomer ACTH(11-24) and the dimer Glu(11-24)2 were also totally devoid of effect on the steroidogenic response to ACTH(1-39) (10(-9)M). In contrast, the (11-24)2Lys conjugate (10(-6)M) significantly decreased ACTH-induced stimulation of corticosterone and aldosterone (-63 and -62%, respectively). The dimeric conjugate of the fragment ACTH(7-24), linked through the C-terminal ends, (7-24)2Lys (10(-6)M), was also completely devoid of effect on basal steroidogenesis but caused a marked decrease of ACTH-evoked corticosterone and aldosterone release (-72 and -80%, respectively). Conversely, infusion of the dimer (1-24)2Lys gave rise to a dose-related stimulation of corticosterone and aldosterone release. The time-course of the steroidogenic response to the dimer was similar to that of ACTH(1-24). The 1-24 conjugate was 70 times less potent than the monomers ACTH(1-24) and ACTH(1-39). These results suggest that amphibian adrenocortical cells contain only one class of ACTH receptor which recognizes the 11-24 domain of ACTH with an affinity which depends on the presence of a strong potentiator segment, located at the N-terminus end of ACTH(1-39). Since the ACTH-dimers are thought to induce cross-linking of the receptors, our results suggest that aggregation of ACTH receptors causes a down-regulation of the receptors.

Adrenal Glands↗
PubMed2004

An allosteric model for transmembrane signaling in bacterial chemotaxis.

Bacteria are able to sense chemical gradients over a wide range of concentrations. However, calculations based on the known number of receptors do not predict such a range unless receptors interact with one another in a cooperative manner. A number of recent experiments support the notion that this remarkable sensitivity in chemotaxis is mediated by localized interactions or crosstalk between neighboring receptors. A number of simple, elegant models have proposed mechanisms for signal integration within receptor clusters. What is a lacking is a model, based on known molecular mechanisms and our accumulated knowledge of chemotaxis, that integrates data from multiple, heterogeneous sources. To address this question, we propose an allosteric mechanism for transmembrane signaling in bacterial chemotaxis based on the "trimer of dimers" model, where three receptor dimers form a stable complex with CheW and CheA. The mechanism is used to integrate a diverse set of experimental data in a consistent framework. The main predictions are: (1) trimers of receptor dimers form the building blocks for the signaling complexes; (2) receptor methylation increases the stability of the active state and retards the inhibition arising from ligand-bound receptors within the signaling complex; (3) trimer of dimer receptor complexes aggregate into clusters through their mutual interactions with CheA and CheW; (4) cooperativity arises from neighboring interaction within these clusters; and (5) cluster size is determined by the concentration of receptors, CheA, and CheW. The model is able to explain a number of seemingly contradictory experiments in a consistent manner and, in the process, explain how bacteria are able to sense chemical gradients over a wide range of concentrations by demonstrating how signals are integrated within the signaling complex.

Allosteric Regulation↗
PubMed2006

Generation of a multimeric form of CD40L with potent immunostimulatory activity using streptavidin as a chaperon.

Effective aggregation of cell surface immune receptors with their ligands is critical in promoting humoral and cellular immune responses. Simulation of these interactions using soluble multimeric ligands having potent adjuvant effects may prove an effective alternative to agonistic antibodies as immunotherapeutics. Multimeric ligands may effectively engage their receptors, leading to aggregation and effective signal transduction. We exploited the structural characteristics of streptavidin (SA) for the generation of multimeric chimeric proteins. Streptavidin forms stable tetramers and oligomers under physiological conditions, and, as such, chimeric molecules with SA are expected to possess similar features. Two chimeric molecules consisting of the extracellular domains of human and mouse CD40L and a modified form of core streptavidin were generated. These proteins form stable oligomers that could only be dissociated into monomers by heating at 100 degrees C, but not 60 degrees C, under denaturing conditions. The chimeric proteins vigorously stimulated B cells, monocytes, and dendritic cells for the production of cytokines and chemokines and upregulation of immunostimulatory molecules. The use of SA as a chaperon presents a novel approach to generate multimeric immunological molecules with potent activities and their use as potential therapeutics for the treatment of cancer and other immune-based disorders.

Animals↗
PubMed1989

Complete structure and expression in transfected cells of high affinity IgE receptor.

The high-affinity receptor for immunoglobulin E, Fc epsilon RI, is found exclusively on mast cells and basophils. When multivalent allergens bind to the receptor-bound IgE, the consequent aggregation of the receptors leads to the release of mediators responsible for allergic symptoms. In rodents Fc epsilon RI is a tetrameric complex of non-covalently attached subunits: one IgE-binding alpha subunit, one beta subunit and a dimer of disulphide-linked gamma subunits. Complementary DNA encoding the alpha and the beta subunits has recently been isolated, but expression of IgE-binding by transfected cells has not yet been achieved. Here we report the cloning of cDNA for the gamma subunit, and propose a model for the alpha beta gamma 2 tetramer which accounts for many of the structural features of the receptor. The rodent receptor on the surface of COS 7 cells was expressed only when the cDNAs for all three subunits were cotransfected. Successful expression of human IgE receptors should now be possible, eventually to permit the detailed analysis of the human IgE-receptor interaction and assist the search for therapeutically effective inhibitors.

Amino Acid Sequence↗
PubMed2002

Ligand binding to somatostatin receptors induces receptor-specific oligomer formation in live cells.

Heptahelical receptors (HHRs) are generally thought to function as monomeric entities. Several HHRs such as somatostatin receptors (SSTRs), however, form homo- and heterooligomers when activated by ligand binding. By using dual fluorescent ligands simultaneously applied to live cells monotransfected with SSTR5 (R5) or SSTR1 (R1), or cotransfected with R5 and R1, we have analyzed the ligand receptor stoichiometry and aggregation states for the three receptor systems by fluorescence resonance energy transfer and fluorescence correlation spectroscopy. Both homo- and heterooligomeric receptors are occupied by two ligand molecules. We find that monomeric, homooligomeric, and heterooligomeric receptor species occur in the same cell cotransfected with two SSTRs, and that oligomerization of SSTRs is regulated by ligand binding by a selective process that is restricted to some (R5) but not other (R1) SSTR subtypes. We propose that induction by ligand of different oligomeric states of SSTRs represents a unique mechanism for generating signaling specificity not only within the SSTR family but more generally in the HHR family.

Animals↗
PubMed1989

Functions associated with the C1q receptor.

Information gathered from various sources indicates that the receptor for human C1q is expressed on a wide range of normal and cultured cell lines. Similarly, the functions mediated by the C1qR are as diverse as the cells that express the molecule. On B lymphocytes, for instance, the C1qR is known to mediate cellular cytotoxicity, induce an anti-proliferative response, inhibit synthesis of IL-1 and modulate the synthesis of Ig, whereas on PMNs and monocytes the C1qR participates in the increase of oxidative metabolism and enhancement of phagocytosis of opsonized target particles. Endothelial cells which form an important barrier between blood and vascular basement membrane use their C1qR to localize C1q bearing immune complexes into the basement membrane and thus initiate an inflammatory response. Human diploid fibroblasts express two types of C1qRs: a low affinity C1qR for the collagen-like domain and a small subpopulation expressing high affinity C1qR which binds to the globular region of C1q. Since the high affinity subpopulation manifests characteristics of fibroblasts participating in wound healing and tissue regeneration, the C1qR on these cells may play an important role at sites of inflammation. Recently, binding of C1q to the C1qR on mouse fibroblasts was shown to induce chemotaxis and K+ conductance activation with an increase in cytosolic Ca2+. Although isolation of C1qR from every cell type has not been accomplished as yet, partial characterization of highly purified C1qR from Raji cells and platelets indicate that at least on these cell types, the structure of the molecule is similar. Both molecules are anionic, single chain glycoproteins of approximately 67-70 kDA and upon reduction on SDS-PAGE migrate to an apparent 80-85 kDa indicative of the presence of disulfide bonds. The sedimentation coefficient was estimated to be 2.4-4.2S for platelet C1qR and 4.2S for that of Raji. Equilibrium isoelectric focusing showed the Raji cell C1qR to migrate with a pI of 5.5-6.0. Because the Raji C1qR reveals two components when analyzed by two dimensional SDS-PAGE, the possibility that the C1qR may be a doublet of two apparent 70 kDa molecules only one of which contains relatively more intrachain disulfide bonds is being raised.

Animals↗
PubMed1987

Effect of thromboxane receptor antagonists on renal artery thrombosis in the cynomolgus monkey.

The effect of the thromboxane (Tx) A2-receptor antagonists SQ 28,668 and SQ 30,741 on platelet function and renal artery thrombosis was studied in the dialurethane-anesthetized cynomolgus monkey. Both antagonists competitively inhibited the aggregation of platelet rich plasma in vitro to arachidonic acid and U-46,619, a Tx-mimetic. SQ 30,741 was 4 to 7 times more potent than SQ 28,668 against either of these agonists. Thrombotic cyclical blood flow reductions (CFRs) were elicited by placing a critical stenosis at a crush injury site on the left renal artery. After allowing 10 consecutive CFRs, of which 95% required shaking of the vessel to restore flow, a single i.v. injection of either SQ 28,668 (1 mg/kg, n = 6), SQ 30,741 (1 mg/kg, n = 8) or vehicle (2 ml of 0.2% Na2CO3 + 10% ethanol, n = 4) was administered. Antithrombotic activity was defined as the spontaneous restoration of flow and was accompanied by a reduction in the rate of flow decline during the CFRs. Spontaneous flow restoration was observed in animals treated with SQ 28,668 (five of six) and SQ 30,741 (six of eight) but not vehicle (zero of four). The rate of flow decline was reduced only with SQ 28,668 (56 +/- 8%) and SQ 30,741 (53 +/- 10%) treatments. The antithrombotic activities of SQ 28,668 and SQ 30,741 lasted 68 +/- 6 and 224 +/- 21 min, respectively. The threshold antithrombotic dose was found to be lower for SQ 30,741 (0.20 +/- 0.03 mg/kg) than SQ 28,668 (0.61 +/- 0.09 mg/kg) in additional experiments.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMedDate not supplied

The 5-hydroxytryptamine2A receptor antagonist sarpogrelate hydrochloride inhibits acute platelet aggregation in injured endothelium.

In this study, the effect of sarpogrelate hydrochloride, a 5-hydroxytryptamine2A receptor antagonist, on platelet aggregation at the site of injured carotid artery endothelium was examined. The rat common carotid artery was clamped for 30 min to induce endothelial injury. Sarpogrelate hydrochloride was administered before and after the injury, and the effects were compared with those in rats receiving sham operation only and those receiving clipping injury but no sarpogrelate hydrochloride. The animals were killed 24 h after the procedure. The common carotid artery was examined by scanning electron microscopy and stained immunochemically for factor VIII. Sarpogrelate hydrochloride treatment was associated with reduced aggregation of platelets on electron microscopy and lower expression of factor VIII at the injured intima. Sarpogrelate hydrochloride has an inhibitory effect on platelet aggregation at the intima in the acute stage after injury, suggesting that this drug may be used to prevent early ischaemic complications after surgical or endovascular arterial intervention.

Animals↗
PubMed2002

A quantitative approach for studying IgE-FcepsilonRI aggregation.

Aggregation of cell surface receptors is a ubiquitous means of initiating signal transduction in many cellular systems. In this manuscript, we describe a combined theoretical and experimental approach based on multiparameter flow cytometry for measuring the time course of ligand induced aggregation of IgE-FcepsilonRI on RBL cells. By fluorescently labeling both the ligand and surface IgE (sIgE), we have developed an assay that permits us to simultaneously measure both occupancy of sIgE combining sites and association of antigen with the cell surface. This allows for a direct calculation of the degree of receptor aggregation present on the cell. By employing new mixing technologies developed for flow cytometry, we are able to look at aggregation in the sub second time domain. To extend our work, we have synthesized a new set of chemically well defined ligands (of valences 1-3) to use as probes in our studies. We show that the magnitude of the cellular response is dramatically increased as the valence of our ligand is raised from two to three.

Animals↗
PubMed2000

Signaling events involved in anti-CD20-induced apoptosis of malignant human B cells.

Anti-CD20 monoclonal antibodies have been successfully employed in the clinical treatment of non-Hodgkin's lymphomas in both unmodified and radio-labeled forms. Previous publications have demonstrated that the antitumor effects of unmodified anti-CD20 mAb are mediated by several mechanisms including antibody-dependent cellular cytotoxicity, complement-mediated cell lysis, and induction of apoptosis by CD20 cross-linking. In this report, we demonstrate induction of apoptosis by three anti-CD20 monoclonal antibodies [1F5, anti-B1, and C2B8 (Rituximab)]. The magnitude of apoptosis induction was greater with the chimeric Rituximab antibody than with the murine 1F5 and anti-B1 antibodies. Apoptosis could be enhanced with any of the antibodies by cross-linking with secondary antibodies (or Fc-receptor-bearing accessory cells). The signaling events involved in anti-CD20-induced apoptosis were investigated, including activation of protein tyrosine kinases, increases in intracellular Ca2+ concentrations, caspase activation, and cleavage of caspase substrates. Our results indicate that anti-CD20-induced apoptosis can be attenuated by PP1, an inhibitor of protein tyrosine kinases Lck and Fyn, chelators of extracellular or intracellular Ca2+, and inhibitors of caspases, suggesting that anti-CD20-induced apoptosis may involve modulation of these signaling molecules. We also demonstrated that varying the expression of Bc1-2 did not affect the magnitude of anti-B1-induced apoptosis, possibly because of the sequestering effects of other Bc1-2 family members, such as Bad. These studies identify several of the signal-transduction events involved in the apoptosis of malignant B cells that transpire following ligation of CD20 by anti-CD20 antibodies in the presence of Fc-receptor-expressing cells or secondary goat anti-(mouse Ig) antibodies and which may contribute to the tumor regressions observed in mouse models and clinical trials.

Amino Acid Chloromethyl Ketones↗
PubMed2000

Ceramide does not inhibit protein kinase C beta-dependent phospholipase D activity stimulated by anti-Fas monoclonal antibody in A20 cells.

We have investigated the roles of ceramide in Fas signalling leading to phospholipase D (PLD) activation in A20 cells. Upon stimulation of Fas signalling by anti-Fas monoclonal antibody, sphingomyelin hydrolysis and activation of PLD were induced. Also, the translocation of protein kinase C (PKC) betaI and betaII and the elevation of diacylglycerol (DAG) content were induced by Fas cross-linking. When phosphatidylcholine-specific phospholipase C (PC-PLC) was inhibited by D609, the Fas-induced changes in PLD activity, DAG content, and PKC translocation were inhibited. In contrast, D609 had no effect on Fas-induced alterations in sphingolipid metabolism, suggesting that changes in ceramide content do not account for Fas-induced PLD activation. Furthermore, C6-ceramide had no effect on Fas-induced PLD activation and PKC translocation. Taken together, these data might suggest that ceramide generated by Fas cross-linking does not affect PKC beta-dependent PLD activity stimulated by anti-Fas monoclonal antibody in A20 cells.

Animals↗
PubMed1996

The tumor necrosis factor receptor 2 signal transducers TRAF2 and c-IAP1 are components of the tumor necrosis factor receptor 1 signaling complex.

The two cell surface receptors for tumor necrosis factor (TNF) interact with a number of intracellular signal transducing proteins. The association of TRADD, a 34-kDa cytoplasmic protein containing a C-terminal death domain, with aggregated TNF receptor 1 (TNF-R1) through their respective death domains leads to NF-kappa B activation and programmed cell death. In contrast, TNF receptor 2 (TNF-R2) interacts with the TNF receptor associated factors 2/1 (TRAF2/TRAF1) heterocomplex, which mediates the recruitment of two cellular inhibitor of apoptosis proteins (c-IAP1 and c-IAP2) to TNF-R2. Here we show that the TNF-R2 signal transducers TRAF2 and c-IAP1 are a part of the TNF-R1 signaling complex. The recruitment of TRAF2 and c-IAP1 to TNF-R1 is TNF-dependent, is mediated by TRADD, and is independent of TNF-R2. These data establish the physiological involvement of TRAF2 and c-IAP1 in TNF-R1 signaling and help provide a molecular explanation for both the overlapping and distinct signals generated by the two TNF receptors.

Antigens, CD↗
PubMed1993

Functional properties of polyclonal antibodies raised against the N-terminus region (residues 9-30) of the follicle-stimulating hormone (FSH) receptor: significance of this receptor region in FSH recognition and signal transduction.

In this study, we raised polyclonal antibodies in rabbits against a synthetic peptide corresponding to a unique region of the FSH receptor, residues 9-30, with no sequence homology to receptors for LH and TSH, and examined their characteristics relevant to receptor function. Binding of [125I]human (h) FSH to membrane-bound receptors was inhibited in a concentration-dependent manner by the anti-FSH receptor-(9-30) peptide antibody. Preimmune serum had no effect. Lineweaver-Burke plot analysis of [125I]hFSH binding to membrane receptors in the presence or absence of antireceptor peptide antibody indicated that the antibody effectively competed with FSH at a hormone-binding site on the receptor. Also, antireceptor peptide antibody, but not preimmune serum, inhibited the ability of FSH to stimulate the conversion of androstenedione to estradiol in cultured immature rat Sertoli cells. Stimulation of estradiol synthesis by Sertoli cells caused by cholera toxin or forskolin (which are known to act through the Gs-protein and catalytic unit of adenylate cyclase, respectively) was not inhibited by antireceptor peptide antibody. Indirect immunofluorescence staining of cultured rat Sertoli cells showed binding of antibody to plasma membrane receptor. No fluorescent staining of receptor was observed when cells were incubated with preimmune serum or antireceptor peptide antibody in the presence of excess receptor-(9-30) peptide or hFSH. These results were consistent with specific labeling of membrane-bound FSH receptors by anti-receptor-(9-30) peptide antibody. When detergent-solubilized membrane preparations from rat Sertoli cells were fractionated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis under nonreducing conditions and then subjected to Western blot analysis, antireceptor peptide antibody, but not preimmune rabbit serum, specifically recognized intact FSH receptor. Although the antireceptor peptide antibody occupied the N-terminus 9-30 epitope region in the FSH receptor, it did not induce postbinding events, such as receptor patching (aggregation), as shown by indirect immunofluorescence staining of rat Sertoli cells and the estradiol response. In contrast, a polyclonal antibody against the FSH holoreceptor capable of interacting with multiple epitopes on the receptor could induce FSH-like effects, such as receptor patching and estradiol response in Sertoli cells. In conclusion, antibody raised against the N-terminus region (9-30) of the FSH receptor recognized intact FSH receptor, inhibited FSH binding, and behaved as an antagonist, suggesting that this N-terminus epitope region of the receptor is involved in hormone binding.(ABSTRACT TRUNCATED AT 400 WORDS)

Amino Acid Sequence↗
PubMed1989

Comparison of sympatho-adrenergic regulation at rest and of the adrenoceptor system in swimmers, long-distance runners, weight lifters, wrestlers and untrained men.

The effects of different physical training regimes on the plasma catecholamine values at rest and the density and responsiveness of adrenergic receptors at rest were investigated. The changes during well-defined training periods of swimmers, long-distance runners, weight lifters and wrestlers were compared with untrained male volunteers. The training of swimmers and long-distance runners, building up endurance, resulted in a significantly lower basal plasma norepinephrine (NE) concentration and a significantly or possibly lower ratio NE:EPI (epinephrine). Both values indicated reduced sympathetic activity and resulted also in a significantly lower beta-receptor density and a higher alpha 2-receptor sensitivity compared with the other groups investigated. However, swimming-specific characteristics provoked labile hypertensive blood pressure regulation with an unchanged heart rate in swimmers. Static training of weight lifters, building up power, also led to a lower NE concentration compared with untrained subjects, whereas beta-receptor density was unchanged and alpha 2-receptor density and sensitivity were decreased. Elevated blood pressure values were observed in weight lifters and swimmers due to a reduced baroreceptor sensitivity. The dynamic training of wrestlers affected only basal heart rate and alpha 2-receptor sensitivity, both of which were decreased. Different kinds of physical training caused various adaptations of the basal activity of the autonomic nervous system in which adrenergic receptors also became adapted. In this context, the stronger adrenergic circulatory component of overall sympathetic activity at rest in swimmers and long-distance runners resulted in lower beta-receptor density, and the reduced noradrenergic component sensitized alpha 2-receptors.

Adenylyl Cyclases↗