The HNK-1 reactive sulfoglucuronyl glycolipids are ligands for L-selectin and P-selectin but not E-selectin.

E-selectin, L-selectin, and P-selectin are related cell adhesion molecules that bind via their lectin domains to sialyl Lewis x and related carbohydrate determinants. Reports have indicated that sulfated glycolipids and polysaccharides also bind selectins. To extend these findings, we compared binding of selectin-IgG chimeras to immobilized sulfated and sialylated glycosphingolipids. E-, L-, and P-selectin chimeras all bound to surfaces absorbed with 2,3-sialyl Lewis x glycolipid or sulfatide (galactosylceramide I3-sulfate) but not to surfaces adsorbed with control sulfated lipids (octadecyl sulfate, sphingosine sulfate). Notably, the L- and P-selectin chimeras but not E-selectin chimera bound to surfaces adsorbed with sulfoglucuronyl glycosphingolipids (SGNL lipids; e.g., IV3 glucuronylneolactotetraosylceramide V3-sulfate). These unusual lipids have been reported as antigenic determinants for monoclonal IgM antibodies produced in patients with neuropathy associated with paraproteinemia and react with the mouse monoclonal antibody HNK-1. Binding of L- and P-selectin chimeras to SGNL lipids was specifically inhibited by appropriate anti-selectin antibodies. While binding of all three selectin chimeras to sialyl Lewis x was blocked by removal of calcium, binding to SGNL lipid was only modestly reduced by EDTA. Chemically desulfated SGNL lipid retained binding activity for L- and P-selectin chimeras, while methyl esterification of the glucuronic acid eliminated binding. We conclude that SGNL lipids, unlike sialyl Lewis x and sulfatides, selectively support L- and P-selectin but not E-selectin chimera binding. The presence of SGNL lipids on brain microvascular endothelium (and other endothelia) may implicate these molecules in leukocyte trafficking to the nervous system and elsewhere.     

Serum concentrations of E-selectin, P-selectin, ICAM-1 and interleukin 6 in acute leukaemia patients with chemotherapy-induced leucopenia and bacterial infections

Summary. Serum concentrations of E-selectin (CD62E), P-selectin (CD62P), ICAM-1 (CD54) and interleukin 6 were investigated in acute leukaemia patients with chemotherapy-induced leucopenia and complicating bacterial infections. Serum concentrations of both E-selectin and P-selectin were decreased in the leucopenic patients without infections when compared with levels before chemotherapy; and serum concentrations of both E-selectin and P-selectin showed a further decrease during complicating bacterial infections. In contrast to the leukaemia patients, previously healthy individuals with meningococcal disease showed markedly elevated serum concentrations of E-selectin and normal levels of P-selectin during infection. Serum concentrations of ICAM-1 and interleukin 6 increased during bacterial infections in the acute leukaemia patients with chemotherapy-induced leucopenia. The alterations in serum concentrations of soluble adhesion molecules and interleukin 6 reversed when clinical signs of bacterial infections resolved during antibiotic therapy. Our results demonstrate that acute leukaemia patients with chemotherapy-induced cytopenia show altered levels of both soluble adhesion molecules and interleukin 6 during complicating bacterial infections.     

Differential regulation of beta 1 and beta 2 integrin avidity by chemoattractants in eosinophils.

The CC chemokines regulated on activation normal T expressed and secreted (RANTES) and monocyte chemotactic protein 3 (MCP-3), and the anaphylatoxin C5a, induce activation, degranulation, chemotaxis, and transendothelial migration of eosinophils. Adhesion assays on purified ligands showed differential regulation of beta 1 and beta 2 integrin avidity in eosinophils. Adhesiveness of VLA-4 (alpha 4 beta 1, CD29/CD49d) for vascular cell adhesion molecule 1 or fibronectin was rapidly increased but subsequently reduced by RANTES, MCP-3, or C5a. The deactivation of VLA-4 lead to cell detachment, whereas phorbol 12-myristate 13-acetate induced sustained activation of VLA-4. In contrast, chemoattractants stimulated a prolonged increase in the adhesiveness of Mac-1 (alpha M beta 2, CD11b/CD18) for intercellular adhesion molecule 1. Inhibition by pertussis toxin confirmed signaling via G protein-coupled receptors. Chemoattractants induced transient, while phorbol 12-myristate 13-acetate induced sustained actin polymerization. Disruption of actin filaments by cytochalasins inhibited increases in avidity of VLA-4 but not of Mac-1. Chemoattractants did not upregulate a Mn2+-inducible beta 1 neoepitope defined by the mAb 9EG7, but induced prolonged expression of a Mac-1 activation epitope recognized by the mAb CBRM1/5. This mAb inhibited chemoattractant-stimulated adhesion of eosinophils to intercellular adhesion molecule 1. Thus, regulation of VLA-4 was dependent on the actin cytoskeleton, whereas conformational changes appeared to be crucial for activation of Mac-1. To our knowledge, this is the first demonstration that physiological agonists, such as chemoattractants, can differentially regulate the avidity of a beta 1 and a beta 2 integrin expressed on the same leukocyte.     

P-selectin glycoprotein ligand 1 and beta2-integrins cooperate in the adhesion of leukocytes to von Willebrand factor

Von Willebrand factor (VWF) is an essential component of hemostasis. However, animal studies using VWF-deficient mice suggest that VWF may also contribute to inflammation. In the present study, we demonstrate that VWF was able to interact with polymorphonuclear leukocytes (PMNs) and monocytes under static and flow conditions. Adhesion under flow was dominated by short-lasting contact with resting PMNs, whereas adhesion of phorbol-12-myristate-13-acetate (PMA)-stimulated PMNs was characterized by firm adhesion. Transient binding of PMNs to VWF appeared to be mediated by P-selectin glycoprotein ligand-1 (PSGL-1). Moreover, recombinant PSGL-1 protein and cell surface-expressed PSGL-1 directly interacted with VWF. As for stable adhesion by PMA-stimulated PMNs, we observed that static adhesion and adhesion under flow were strongly inhibited (greater than 75%) by neutrophil-inhibitory factor, an inhibitor of beta2-integrin function. In addition, the isolated I-domain of alphaMbeta2 bound to VWF, and cell lines expressing alphaLbeta2 or alphaXbeta2 adhered efficiently to VWF. Taken together, our data showed that VWF can function as an adhesive surface for various leukocyte subsets (monocytes, PMNs). Analogous to VWF-platelet interaction, VWF provided binding sites for leukocyte receptors involved in rolling (PSGL-1) and stable (beta2-integrins) adhesion. VWF is unique in its intrinsic capacity to combine the rolling and the stable adhesion step in the interaction with leukocytes.     

Circulating levels of soluble E-selectin, P-selectin and intercellular adhesion molecule-1 in patients with juvenile idiopathic arthritis

OBJECTIVE: To measure circulating levels of soluble E-selectin (sE-selectin), sP-selectin, and soluble intercellular adhesion molecule-1 (sICAM-1) in patients with active juvenile idiopathic arthritides (JIA), and to evaluate their correlation with disease activity variables and cytokine levels. METHODS: Serum levels of sE-selectin, sP-selectin, and sICAM-1 were measured by ELISA in 42 patients with JIA and in 15 healthy controls. RESULTS: Circulating levels of sE-selectin and sICAM-1, but not sP-selectin, were significantly elevated in patients with active systemic JIA. In patients with active polyarticular or pauciarticular JIA serum levels of sE-selectin. sP-selectin, and sICAM-1 were comparable to those of controls. In patients with systemic JIA, levels of sE-selectin and sICAM-1 were significantly correlated with levels of soluble tumor necrosis factor receptor 2 (sTNFR2), but not with those of interleukin 6 (IL-6) or IL-1beta. CONCLUSION: Patients with active systemic JIA have elevated circulating levels of sE-selectin and sICAM-1. The correlation with sTNFR2, together with previous data on the TNF system in systemic JIA. suggests that TNF activated endothelial cells are the source of sE-selectin and sICAM-1 in this disease.     

Cyclooxygenase 2 pathway mediates IL-1beta regulation of IL-1alpha, -1beta,and IL-6 mRNA levels in Leydig cell progenitors

Prostanoids are arachidonic acid (AA) metabolites derived from the cyclooxygenase (COX1 and COX2 isozymes) pathway and are involved in signal transduction pathways activated by distinct ILs. Although COX1 is the constitutive isoform of COX, IL-1beta is a potent inducer of COX2 expression in distinct cell types. This study was designed to determine whether cyclooxygenases could mediate endogenous cytokine regulation in rat progenitor Leydig cells. COX and IL (IL-1alpha, IL-1beta, and IL-6) mRNAs were measured by PCR and real-time PCR analyses, respectively. COX function was assessed using COX activity inhibitors: indomethacin (INDO; COX1 and COX2 inhibitor) and NS-398 (COX2 selective inhibitor). Our data indicate that endogenous progenitor COX1 mRNA levels are low and are not regulated by IL-1beta. In contrast, COX2 mRNA is induced by IL-1beta at 6, 9, and 24 h. IL-1beta induction of IL mRNAs was in part significantly impaired in the presence of INDO or NS-398. Among the prostanoids tested, prostaglandin E(2) (PGE(2)), PGF(2alpha), and carbaprostacyclin reversed the INDO inhibition of IL production. PGs alone have no (IL-1alpha and IL-1beta) or a modest (IL-6) effect on IL mRNA levels. PGE(2), PGF(2alpha), and PGI(2) measurements show that IL-1beta treatment significantly increases progenitor Leydig cell production of these PGs. Taken together, our data demonstrate that this COX2 cascade is a regulator of cytokines in Leydig progenitors.     

Differential adrenergic regulation of beta 1- and beta 3-adrenoreceptor messenger ribonucleic acids in adipose tissues.

The levels of beta 1- and beta 3-adrenoreceptor mRNAs in several rat tissues were determined simultaneously with a sensitive nuclease protection assay. The beta 1-receptor gene was expressed to varying degrees in most tissues examined. By contrast, high levels of beta 3-receptor mRNA were only found in brown and white adipose tissues, indicating that beta 3-receptor gene expression is essentially adipose tissue specific. Surgical sympathectomy of interscapular brown adipose tissue increased beta 3-receptor mRNA levels by 2.4-fold, but did not affect beta 1-receptor mRNA levels. Exposure of rats to 4 C, which increases sympathetic nerve stimulation of IBAT, reduced beta 3-receptor mRNA levels in intact tissue but did not affect the denervation-induced increase in beta 3-receptor mRNA. Acute treatment of rats with norepinephrine greatly reduced beta 3 mRNA levels in both white and brown adipose tissues, but did not alter beta 1-receptor mRNA levels. These results indicate that beta 1- and beta 3-receptor mRNAs are differentially regulated and that norepinephrine released from sympathetic nerves is an important inhibitory regulator of beta 3-receptor mRNA levels. Injections of the beta-receptor agonist isoproterenol and the beta 3-receptor agonist BRL 26830 each reduced beta 3-receptor mRNA in brown and white fat, whereas injections of glucagon reduced beta 3-receptor mRNA in brown fat only. These data indicate that while stimulation of beta 3-receptors is sufficient to down-regulate beta 3 mRNA, other receptors that stimulate adenylyl cyclase have the same effect. Finally, the agonist-induced down-regulation of beta 3-receptor mRNA was associated with a reduction in beta 3-receptor activation of adenylyl cyclase in white adipose tissue.     

Differential requirements for Alix and ESCRT-III in cytokinesis and HIV-1 release

The ESCRT machinery functions in topologically equivalent membrane fission events, namely multivesicular body formation, the terminal stages of cytokinesis and HIV-1 release. Here, we show that the ESCRT-III-binding protein Alix is recruited to the midbody of dividing cells through binding Cep55 via an evolutionarily conserved peptide. Disruption of Cep55/Alix/ESCRT-III interactions causes formation of aberrant midbodies and cytokinetic failure, demonstrating an essential role for these proteins in midbody morphology and cell division. We also show that the C terminus of Alix encodes a multimerization activity that is essential for its function in Alix-dependent HIV-1 release and for interaction with Tsg101. Last, we demonstrate that overexpression of Chmp4b and Chmp4c differentially inhibits HIV-1 release and cytokinesis, suggesting possible reasons for gene expansion within the mammalian Class E VPS pathway.     

Biological determinants of serum ICAM-1, E-selectin, P-selectin and L-selectin levels in healthy subjects: the Stanislas study

BACKGROUND: Circulating levels of adhesion molecules increase in various inflammation-related diseases, such as atherosclerosis. However, data about factors influencing their concentrations in physiological conditions are scarce. METHODS: We have studied the determinants of serum levels of intercellular adhesion molecule-1 (ICAM-1), E-selectin, P-selectin and L-selectin in a sample of healthy individuals: 303 children (4-17 years) and 493 adults (18-55 years). The concentrations of these molecules have been measured by enzyme-linked immunosorbant assay. RESULTS: As far as the children are concerned, a decrease in the levels of ICAM-1, E-selectin, P-selectin and L-selectin has been noticed for both boys and girls aged 4-17 years, without any difference between genders. For the adults, no age-related variation has been found for the ICAM-1, E-selectin and P-selectin levels, while the L-selectin level decreased until 55 years old. In the adult group, no sex-related difference in the concentrations of ICAM-1, E-selectin and L-selectin has been seen. As to the P-selectin level, men had significantly higher levels than women. Multiple regression analysis showed that smoking, homeostasis model assessment (HOMA) index, aspartate aminotransferase (AST), alkaline phosphatase (ALP) and C-reactive protein (CRP) were significant positive determinants of the ICAM-1 concentration, whereas age and apo AI were negative ones. The E-selectin level was positively associated with body mass index (BMI), leukocyte, platelet and erythrocyte counts, glucose, ALP and tumor necrosis factor-alpha (TNF-alpha), and negatively related to the use of oral contraceptive (OC). Positive determinants of the P-selectin concentration were leukocyte, platelet and erythrocyte counts, whereas sex, the use of oral contraceptive, glucose and TNF-alpha were negative determinants of P-selectin. Only two determinants have been noticed for the concentration of serum L-selectin: age, which was negatively correlated, and leukocyte count, which was positively associated. CONCLUSION: Our study contributes to the understanding of the regulation of adhesion molecules in physiological conditions.     

Involvement of beta1- and beta2- but not beta3-adrenoceptor activation in adrenergic PYY secretion from the isolated colon

The secretion of PYY by endocrine L cells of the terminal gut is under the control of nutrients, the autonomic nervous system and hormones. Catecholamines, and the non-specific beta-adrenergic agonist isoproterenol induce PYY secretion from rat isolated colon or ileum. Because beta3-adrenergic receptors now appear to mediate many of the effects of catecholamines in the gastrointestinal tract, we investigated the involvement of beta1-, beta2-, and beta3-adrenoceptor stimulation in PYY secretion from the isolated, vascularly perfused rat colon. Infusion of 10(-6) M isoproterenol induced a transient increase in PYY secretion (from 36+/-4 to 87+/-20 fmol/2 min; n=7, P<0.05), that was abolished by a previous infusion of the beta1- and beta2-adrenergic blocker (and partial beta3-agonist) alprenolol (10(-6) M). The beta1-adrenergic agonist dobutamine and the beta-2 agonist terbutaline also (both at 10(-5) M) significantly stimulated PYY secretion, from 29+/-1 to 79+/-12 fmol/2 min and from 19+/-1 to 73+/-13 fmol/2 min respectively (n=7, P<0.05). Neither of the beta3-adrenergic agonists tested (BRL 37 344 (10(-5), 10(-6) M) and SR 58 611A (10(-6) M)) significantly stimulated PYY secretion, thus confirming the exclusive involvement of beta1- and beta2-receptors in beta-adrenergic agonist induced hormone secretion.     

E-selectin, but not P-selectin, is required for development of adjuvant-induced arthritis in the rat.

OBJECTIVE: To determine the role of the endothelial cell adhesion molecules E- and P-selectin in the development and severity of adjuvant-induced arthritis (AIA) in the rat. METHODS: Lewis rats were immunized subcutaneously with Mycobacterium butyricum (Mb), and blocking monoclonal antibodies (mAb) to rat E- and P-selectin were administered. Clinical score, radiolabeled (51Cr and 111In) blood polymorphonuclear leukocyte (PMN) and monocyte migration to joints, and histologic features were monitored. RESULTS: When mAb treatment was started on day 5 postimmunization with Mb (preclinical stage), development of AIA was significantly (P < 0.01) inhibited by mAb to E- but not to P-selectin (mean score on day 14 control 10.2, anti-E 2.8, anti-P 9.1). This was associated with markedly decreased migration (by 66-94%) of PMN and monocytes to arthritic joints and diminished cartilage degradation. When treatment was delayed until animals showed signs of arthritis (day 10 postimmunization), only a marginal and variable effect was observed as compared with blockade during the preclinical (day 5) stage. E-selectin blockade on day 5 and day 7 postimmunization resulted in inhibition of antigen-dependent T cell-mediated inflammation, since it decreased T cell migration to sites of dermal-delayed hypersensitivity induced by Mb without affecting migration to concanavalin A or cytokines. The proliferative response of T cells to Mb in vitro was not altered. CONCLUSION: E-selectin plays an important role early in the development of AIA. This adhesion molecule may contribute to the migration of antigen-reactive T cells to peripheral tissues, including the joints where T cells initiate the arthritis.     

Quantitative autoradiography of beta 1- and beta 2-adrenergic receptors in rat brain.

We have used quantitative autoradiography to localize in rat brain beta 1- and beta 2-adrenergic receptors. These receptors were labeled in vitro with 125I-labeled pindolol, an antagonist of beta-adrenergic receptors that binds nonselectively to both beta 1 and beta 2 subtypes. The selective inhibition of 125I-labeled pindolol binding with specific antagonists of beta 1 and beta 2 receptors allowed the visualization of beta-adrenergic receptor subtypes. High levels of beta 1 receptors were observed in the cingulate cortex, layers I and II of the cerebral cortex, the hippocampus, the Islands of Calleja, and the gelatinosus, mediodorsal, and ventral nuclei of the thalamus. High levels of beta 2 receptors were found in the molecular layer of the cerebellum, over pia mater, and in the central, paraventricular, and caudal lateral posterior thalamic nuclei. Approximately equal levels of beta 1 and beta 2 receptors occurred in the substantia nigra, the olfactory tubercle, layer IV of the cerebral cortex, the medial preoptic nucleus, and all nuclei of the medulla. The pronounced differences in the ratio of beta 1 to beta 2 receptors among brain regions suggests that the subtypes of beta-adrenergic receptors may play different roles in neuronal function.     

Localization of beta 1- and beta 2-adrenergic receptors in rat kidney by autoradiography

beta-Adrenergic receptor subtypes were localized and differentiated in rat kidney slices by in vitro autoradiography using the nonselective beta-antagonist [125I]iodocyanopindolol in the presence of the selective agents betaxolol (beta 1) and zinterol (beta 2). [125I]Iodocyanopindolol binding to kidney sections in the presence of these agents could be differentiated into high- and low-affinity components as predicted by the subtype selectivity of the compounds. Autoradiography revealed that: beta-adrenergic receptors were highly concentrated within the renal cortex, especially in glomeruli and juxtaglomerular granule cells, and to tubular sites in the cortex and medulla; [125I]iodocyanopindolol labeling of the juxtaglomeruluar granule cells was abolished at lower concentrations (10(-8) M) of betaxolol than was the labeling of glomeruli (10(-6)--10(-4) M); and zinterol had little effect on labeling of juxtaglomerular granule cells and glomeruli unless high concentrations were used, whereas the tubular labeling in medulla was much more sensitive to incubation with zinterol. These results indicate that beta 1- and beta 2-adrenergic receptor subtypes are differentially distributed within the kidney: beta 1, predominantly contained in juxtaglomerular granule cells and glomeruli, and beta 2, predominantly in medullary tubules. In vitro autoradiography provides a useful means to examine different receptor populations in discrete tissue areas.     

Cardiovascular and metabolic alterations in mice lacking beta1- and beta2-adrenergic receptors

Targeted disruption of murine beta-adrenergic receptor (beta-AR) genes has helped to clarify the role of specific beta-AR subtypes in regulating cardiovascular development and function. In the mouse, the beta1-AR is primarily responsible for sympathetic regulation of both cardiac chronotropy and inotropy. In contrast, all three beta-ARs play a role in regulating peripheral vascular tone. The impact of ablation of both beta1- and beta2-ARs on cardiac development and on resting cardiovascular and metabolic parameters is remarkably minimal. However, exercise stress reveals additional important contributions of beta1- and beta2-ARs to cardiovascular performance.     

IL-32 synergizes with nucleotide oligomerization domain (NOD) 1 and NOD2 ligands for IL-1beta and IL-6 production through a caspase 1-dependent mechanism

The activation of innate immunity requires the amplification of signals induced by pattern-recognition receptors for bacterial products. We have investigated the role of the newly described cytokine IL-32 in the amplification of cytokine production induced by the two most clinically relevant families of microbial receptors, the cell-surface Toll-like receptors (TLRs) and the intracellular nuclear oligomerization domain (NOD) receptor family. IL-32 synergized with the NOD1- and NOD2-specific muropeptides of peptidoglycans for the release of IL-1beta and IL-6 (a 3- to 10-fold increase). In contrast, IL-32 did not influence the cytokine production induced via TLRs. The synergistic effect of IL-32 and synthetic muramyl dipeptide (MDP) on cytokine production was absent in the cells of patients with Crohn's disease bearing the NOD2 frameshift mutation 3020insC, demonstrating that the IL-32/MDP synergism depends on NOD2. This in vitro synergism between IL-32 and NOD2 ligands was consistent with a marked constitutive expression of IL-32 in human colon epithelial tissue. In addition, the potentiating effect of IL-32 on the cytokine production induced by the synthetic muropeptide FK-156 was absent in NOD1-deficient macrophages, supporting the interaction between IL-32 and NOD1 pathways. When specific caspase inhibitors were used, the synergism between IL-32 and MDP/NOD2 depended on the activation of caspase 1. Only additive effects of IL-32 and muropeptides were observed for TNF-alpha production. The modulation of intracellular NOD2 pathways by IL-32, but not cell-surface TLRs, and the marked expression of IL-32 in colon mucosa suggest a role of IL-32 in the pathogenesis of Crohn's disease.     

Fringe-mediated extension of O-linked fucose in the ligand-binding region of Notch1 increases binding to mammalian Notch ligands

The Notch signaling pathway is essential for many aspects of development, cell fate determination, and tissue homeostasis. Notch signaling can be modulated by posttranslational modifications to the Notch receptor, which are known to alter both ligand binding and receptor activation. We have modified the ligand-binding region (EGF domains 11–13) of human Notch1 (hN1) with O-fucose and O-glucose glycans and shown by flow cytometry and surface plasmon resonance that the Fringe-catalyzed addition of GlcNAc to the O-fucose at T466 in EGF12 substantially increases binding to Jagged1 and Delta-like 1 (DLL1) ligands. We have subsequently determined the crystal structures of EGF domains 11–13 of hN1 modified with either the O-fucose monosaccharide or the GlcNAc–fucose disaccharide at T466 of EGF12 and observed no change in backbone structure for each variant. Collectively, these data demonstrate a role for GlcNAc in modulating the ligand-binding site in hN1 EGF12, resulting in an increased affinity of this region for ligands Jagged1 and DLL1. We propose that this finding explains the Fringe-catalyzed enhancement of Notch–Delta signaling observed in flies and humans, but suggest that the inhibitory effect of Fringe on Jagged/Serrate mediated signaling involves other regions of Notch.The Notch signaling pathway is universally conserved among all metazoan species and is involved in many aspects of cell fate determination, tissue patterning, and homeostasis (1). Notch (N) receptors are large single-pass transmembrane glycoproteins that undergo cleavage by a furin-like convertase before being targeted to the cell surface as a heterodimer (2, 3). The Notch extracellular domain (N_ECD) contains up to 36 tandem epidermal growth factor-like (EGF) modules, and EGF 11–12 in Drosophila (d)Notch and human (h)N1 (4, 5) are required for Notch to bind to the Delta, Serrate, Lag-2 (DSL) domain of a ligand molecule on an adjacent cell (6, 7). Drosophila has a single Notch receptor and two ligands, Delta and Serrate. Mammalian species contain four Notch homologs (Notch1–4) and five ligands [Jagged1 and 2 and Delta-like ligand (DLL) 1, 3, and 4], which are also expressed as large single-pass transmembrane proteins (8). Endocytosis of the receptor–ligand complex induces a conformational change in the negative regulatory region of the Notch receptor, which is cleaved by ADAM 10 (9) and the γ-secretase complex (10, 11). This process removes the N_ECD from the surface of the Notch-expressing cell and releases the Notch intracellular domain (N_ICD) from the membrane (12). N_ICD translocates to the nucleus, where it can activate expression of the HES/HEY family of genes (13).Many of the EGF modules in the N_ECD are modified by O-fucose and/or O-glucose glycans (14–16). O-fucose is added to Ser or Thr residues in the consensus sequence C²-X-X-X-X-(S/T)-C³ (14, 17) by protein O-fucosyltransferase (Pofut)-1 in mammals (Ofut1 in flies) and is essential for optimal Notch signaling in flies and mice (18, 19). Similarly, O-glucose can also be added to a Ser in the consensus C¹-X-S-X-(P/A)-C² by protein O-glucosyltransferase (Poglut). O-glucosylation is also essential for Notch signaling in mouse and Drosophila (20, 21). The molecular mechanism for the effects of these sugars on Notch activity is not yet clear. The presence of O-fucose can enhance ligand binding by mammalian Notch receptors (22, 23), whereas O-glucose does not appear to affect ligand binding but a subsequent step before receptor cleavage (20, 21).Many of the O-fucose monosaccharides on Notch can be extended with GlcNAc by a single Fringe enzyme in Drosophila and by any of three Fringe enzymes—Lunatic fringe (Lfng), Manic fringe (Mfng), or Radical fringe (Rfng) in mammalian organisms (24), each of which affects Notch activity in slightly different ways (17, 25–27). The simplest case appears in flies, where Fringe modification has a positive effect on Delta-mediated signaling and a negative effect on Serrate-mediated signaling. Prior studies have reported variable, and sometimes contradictory, effects of mammalian Fringes on Notch signaling (22, 26–30) (SI Appendix, Table S1). This variability may be the result of the different cell systems used in these studies, which could cause cell-based variations in the extent of Notch glycosylation. For instance, it is known that the GlcNAc–fucose disaccharide can be further extended with galactose and sialic acid to produce the mature tetrasaccharide: Siaα2–3/6Galβ1–4GlcNAcβ1–3fucose, and this subsequent extension is known to further modulate Notch signaling (30, 31). The molecular basis for the effects of Fringe on Notch signaling is unknown.The core ligand-binding site in hN1 is located on the central β-sheet of EGF12 (32), directly adjacent to the threonine residue that forms part of the O-fucosylation consensus sequence within this domain (T466). Sugar modifications at this position therefore have the potential to directly or indirectly affect formation of the Notch ligand complex through intermolecular or intramolecular effects. Based on this observation, we have modified prokaryotically expressed and refolded hN1_11–13 with various O-fucose and -glucose sugars in vitro, and assayed the effects of these modifications on binding to a variety of different mammalian Notch ligands using a flow cytometry-based assay and by surface plasmon resonance (SPR). In parallel, we have determined the crystal structures of hN1_11–13 modified with both the O-fucose monosaccharide and disaccharide at this position. Collectively, these data demonstrate that the GlcNAc addition by Fringe to O-fucosylated EGF12 substantially enhances the affinity of this region for the ligands Jagged1 and DLL1 without altering the backbone structure of EGF12. Importantly, these data provide a plausible molecular explanation for the positive effect of Fringe on Delta-mediated signaling.     

Thyroid hormone-induced up-regulation of beta1- and beta2-adrenergic receptors during aging

Strong experimental evidence suggests that thyroid hormones influence beta-adrenoceptor regulation. The question arises whether in tissues like mouse brain cortex bearing both beta1- and beta2-subpopulations, these receptor subtypes may be regulated independently, as demonstrated after some drug stimulations. An eventual influence of aging on triiodothyronine (T(3))-induced subpopulation response is also worth studying, because of the decrease of receptor density in old animals observed only in the beta1-type. These points have been addressed in the present paper, performing experiments on young and old mice stimulated with a single injection of T(3). The response has been assayed as rapidly as 15 min after T(3) treatment. Results on untreated control mice confirm previous findings showing that the decrease of receptor density is in charge only of the beta1-subtype. T(3) stimulation induces up-regulation of both beta1- and beta2-adrenoceptors in young animals. The response shows no impairment with aging, as in both receptor subtypes a significant increase can be demonstrate. Due to the very short time of response, and unmasking mechanism can be hypothesized for receptor up-regulation, though an increase of protein synthesis cannot be excluded.     

Characterization of beta 1- and beta 2-adrenoceptor subtypes in the rat atrioventricular node by quantitative autoradiography

We characterized beta-adrenoceptor subtypes in the atrioventricular node of the rat heart by quantitative autoradiography. Consecutive 16-microns-thick sections from single rat hearts containing the atrioventricular node were incubated with increasing concentrations of [125I]iodocyanopindolol. After exposure to [3H]Ultrofilm, optical densities corresponding to the atrioventricular node were determined by computerized densitometry after comparison with [125I]standards. The computer program LIGAND was used for analysis of receptor subtypes. Delineation of beta-adrenoceptor subtypes was achieved by incubating consecutive tissue sections with 50 pM [125I]iodocyanopindolol in the presence of increasing concentrations of the beta 1-selective antagonist atenolol or the beta 2-selective antagonist ICI 118,551. The atrioventricular node contains a higher concentration of beta-adrenoceptors than the adjacent interventricular septum. We estimated that the proportions of beta 1- and beta 2-adrenoceptors in the atrioventricular node were about 56% and 44% of the total binding capacity respectively.     

Densitometric analysis of beta 1- and beta 2-adrenoceptors in guinea-pig atrioventricular conducting system.

Quantitative autoradiography was used to determine the densities of beta 1- and beta 2-adrenoceptors in the atrioventricular conducting system in guinea-pig. (-)[125I]Cyanopindolol (CYP) was used to label beta 1- and beta 2-adrenoceptors in the absence or presence of the beta 1-adrenoceptor selective antagonist CGP 20712A (100 nM) or the beta 2-adrenoceptor selective antagonist ICI 118,551 (70 nM) or the non-selective beta-adrenoceptor antagonist (-)-propranolol (1 microM). Protein in discrete anatomical regions was determined using a densitometric method based on the dye Coomassie brilliant blue G. In the atrioventricular conducting system the proportion of beta 2-adrenoceptors determined by inhibition of total (-)[125I]-CYP binding by CGP 20712A (100 nM) ranged from 32.5% (atrioventricular node) to 48.7% (left bundle branch). In the atrioventricular node (16.8 fmol/mg protein), bundle of His (12.1 fmol/mg protein), right (17.4 fmol/mg protein) and left (21.1 fmol/mg protein) bundle branches and Purkinje cells there was a higher density of beta 2-adrenoceptors than in the interventricular septum (8.4 fmol/mg protein) and right atria (8.3 fmol/mg protein). The medial smooth muscle of the aorta, aortic valve, adventitia of the aorta, nerve tissue, tricuspid and mitral valves contained only beta 2-adrenoceptors. It is speculated that the use of beta-adrenoceptor antagonists to control cardiac arrhythmias involving a defect in conduction in the atrioventricular node should take into consideration both beta 1- and beta 2-adrenoceptors.