Functional distribution and further characterization of human endothelial ligand for cellular L-selectin.

In the present study we examine the functional distribution of the human endothelial L-selectin ligand, which determines the sites of extravasation of L-selectin-positive cells. A murine cell line transfected with human L-selectin adhered preferentially to the high endothelial venules (HEV) of human peripheral lymph nodes compared to the HEV of mucosal lymphoid tissues (mean of 0.83 compared to a mean of 0.07 cells per HEV respectively). In addition, an antibody against L-selectin differentially inhibited the adhesion of human lymphocytes to peripheral lymphoid tissue versus mucosal lymphoid tissue HEV (mean 41 and 5% inhibition respectively). Although both sulfoglucuronyl-containing glycolipids and sialyl-Lewis X have been proposed as endothelial ligands for L-selectin, an antibody against the former did not bind to peripheral lymph node endothelium, and an antibody against the latter did not block adhesion of L-selectin-expressing cells. The enzyme O-sialoglycoprotein endopeptidase caused up to an 84% reduction in L-selectin-dependent binding, indicating that sialylated glycoproteins containing O-linked glycans are essential for a large majority of adhesion via L-selectin.     

Specificities of N-acetylglucosamine-6-O-sulfotransferases in relation to L-selectin ligand synthesis and tumor-associated enzyme expression.

N-Acetylglucosamine-6-O-sulfotransferase (GlcNAc6ST) catalyzes the transfer of sulfate from adenosine 3'-phosphate,5'-phosphosulfate to the C-6 position of the non-reducing GlcNAc. Three human GlcNAc6STs, namely GlcNAc6ST-1, GlcNAc6ST-2 (HEC-GlcNAc6ST), and GlcNAc6ST-3 (I-GlcNAc6ST), were produced as fusion proteins to protein A, and their substrate specificities as well as their enzymological properties were determined. Both GlcNAc6ST-1 and GlcNAc6ST-2 efficiently utilized the following oligosaccharide structures as acceptors: GlcNAcbeta1-6[Galbeta1-3]GalNAc-pNP (core 2), GlcNAcbeta1-6ManOMe, and GlcNAcbeta1-2Man. The ratios of activities to these substrates were not significantly different between the two enzymes. However, GlcNAc6ST-2 but not GlcNAc6ST-1 acted on core 3 of GlcNAcbeta1-3GalNAc-pNP. GlcNAc6ST-3 used only the core 2 structure among the above mentioned oligosaccharide structures. The ability of GlcNAc6ST-1 to sulfate core 2 structure as efficiently as GlcNAc6ST-2 is consistent with the view that GlcNAc6ST-1 is also involved in the synthesis of l-selectin ligand. Indeed, cells doubly transfected with GlcNAc6ST-1 and fucosyltransferase VII cDNAs supported the rolling of L-selectin-expressing cells. The activity of GlcNAc6ST-2 on core 3 and its expression in mucinous adenocarcinoma suggested that this enzyme corresponds to the sulfotransferase, which is specifically expressed in mucinous adenocarcinoma (Seko, A., Sumiya, J., Yonezawa, S., Nagata, K., and Yamashita, K. (2000) Glycobiology 10, 919-929).     

6-O-sulfo de-N-acetylsialyl Lewis X as a novel high-affinity ligand for human L-selectin: total synthesis and structural characterization

Total synthesis and structural characterization of a novel 6-O-sulfo de-N-acetylsialyl Lewis X, which was originally discovered as a minor by-product of the parent 6-O-sulfo N-acetylsialyl Lewis X, a high-affinity endogenous ligand for human L-selectin, are described. The total synthesis has been achieved by a highly efficient, regio- and alpha-stereoselective glycosylation of N-trifluoroacetylneuraminic acid, selective protections of the 3- and 6-hydroxyl groups of N-acetylglucosamine that undergo fucosylation and sulfation, and construction of the glycolipid structure containing a ceramide. The structure of 6-O-sulfo de-N-acetylsialyl Lewis X ganglioside was characterized by fast atom bombardment mass spectrometry (FAB-MS).     

A unique PPARgamma ligand with potent insulin-sensitizing yet weak adipogenic activity.

FMOC-L-Leucine (F-L-Leu) is a chemically distinct PPARgamma ligand. Two molecules of F-L-Leu bind to the ligand binding domain of a single PPARgamma molecule, making its mode of receptor interaction distinct from that of other nuclear receptor ligands. F-L-Leu induces a particular allosteric configuration of PPARgamma, resulting in differential cofactor recruitment and translating in distinct pharmacological properties. F-L-Leu activates PPARgamma with a lower potency, but a similar maximal efficacy, than rosiglitazone. The particular PPARgamma configuration induced by F-L-Leu leads to a modified pattern of target gene activation. F-L-Leu improves insulin sensitivity in normal, diet-induced glucose-intolerant, and in diabetic db/db mice, yet it has a lower adipogenic activity. These biological effects suggest that F-L-Leu is a selective PPARgamma modulator that activates some (insulin sensitization), but not all (adipogenesis), PPARgamma-signaling pathways.     

Energetics of ligand and inhibitor interactions with acetylcholinesterase

Acetylcholinesterase (AChE, EC 3.1.1.7) from Electrophorus electricus, purified by affinity chromatography to a specific activity of 7000-10,000 U/mg protein, was studied at 27 degrees C in conduction-type microcalorimeters for the heats of reaction, with the subsite-specific cationic ligands edrophonium and propidium and with the irreversible inhibitor diisopropylfluorophosphate (DFP), in an ion-free aqueous medium. Edrophonium and propidium, each at 0.5 x 10(-5) M, yielded reaction heats of +3.2 and -1.5 kcal/mol (1 kcal = 4.184 J) respectively, with 1.3 x 10(-5) M AChE active sites. DFP (1.3 x 10(-5) M) reacted exothermically yielding -0.5 kcal/mol at stoichiometric level with AchE active sites. Circular dichroic spectra showed that a ternary complex of AChE (6.5 x 10(-7) M active sites) and the two ligands (each at 1 x 10(-3) M) in 1 mM Tris-HCl buffer (pH 8.0) had a positive Cotton effect at 235 nm. Neither DFP nor phosphoric acid 2,2-dichloroethenyl dimethyl ester (DDVP) caused any appreciable change. DFP-AChE, however, behaved like a normal enzyme in showing a positive Cotton effect in association with the two ligands. DDVP-AChE showed an increase in negative ellipticity at 287 nm in the presence of the two ligands. Another cationic ligand, d-tubocurarine, when present together with edrophonium, increased negative ellipticity at 302 nm and blue-shifted a 265-nm peak of the normal AChE. DFP interactions with AChE appear to be energetically different from those of edrophonium, the latter of which is believed to associate with the acetylcholine-binding subsite.     

Identification of potent and novel small-molecule inhibitors of caspase-3

The design and synthesis of a series of novel, reversible, small molecule inhibitors of caspase-3 are described.     

Synthesis and anticancer activity of novel bisindolylhydroxymaleimide derivatives with potent GSK-3 kinase inhibition

Synthesis and biological evaluation of a series of novel indole derivatives as anticancer agents is described. A bisindolylmaleimide template has been derived as a versatile pharmacophore with which to pursue chemical diversification. Starting from maleimide, the introduction of an oxygen to the headgroup (hydroxymaleimide) was initially investigated and the bioactivity assessed by screening of kinase inhibitory activity, identifying substituent derived selectivity. Extension of the hydroxymaleimide template to incorporate substitution of the indole nitrogens was next completed and assessed again by kinase inhibition identifying unique selectivity patterns with respect to GSK-3 and CDK kinases. Subsequently, the anticancer activity of bisindolylmaleimides were assessed using the NCI-60 cell screen, disclosing the discovery of growth inhibitory profiles towards a number of cell lines, such as SNB-75 CNS cancer, A498 and UO-31 renal, MDA MB435 melanoma and a panel of leukemia cell lines. The potential for selective kinase inhibition by modulation of this template is evident and will inform future selective clinical candidates.     

Sialomucin CD34 is the major L-selectin ligand in human tonsil high endothelial venules

Peripheral node addressin (PNAd) is a complex mixture of glycoproteins with L-selectin ligand activity that functions in lymphocyte homing. We have investigated the contribution of the sialomucin CD34 relative to other components of PNAd in lymphocyte tethering and rolling in in vitro laminar flow assays. PNAd was isolated with MECA-79 mAb-Sepharose from tonsillar stroma, and the CD34 component (PNAd,CD34+) and CD34- negative component (PNAd,CD34-) separated on CD34 mAb-Sepharose. Lymphocytes on the PNAd,CD34- fraction tether less efficiently, roll faster and are less resistant to shear detachment than on PNAd. The PNAd,CD34+ fraction constitutes about half the total functional activity. These studies show that CD34 is a major functional component of PNAd. Ligand activity in both the PNAd,CD34+ and PNAd,CD34- fractions is expressed on mucin-like domains, as shown with O- sialoglycoprotease. The CD34 component of PNAd has about four times higher tethering efficiency than total tonsillar CD34. CD34 from spleen shows no lymphocyte tethering. Although less efficient than the PNAd,CD34+ fraction from tonsil, CD34 from the KG1a hematopoietic cell line is functionally active as an L-selectin ligand despite lack of reactivity with MECA-79 mAb, which binds to a sulfation-dependent epitope. All four forms of CD34 are active in binding to E-selectin. KG1a CD34 but not spleen CD34 are active as L-selectin ligands, yet both lack MECA-79 reactivity and possess E-selectin ligand activity. This suggests that L-selectin ligands and E-selectin ligands differ in more respects than presence of the MECA-79 epitope.     

Intra- and intertissular cytomictic interactions in the microsporogenesis of mono- and dicotyledonous plants

Comparative cytological analysis of intra- and intertissular cytomictic interactions in the microsporogenesis of mono- and dicotyledonous plants has been performed for two cellular systems: the microsporocytes and the tapetum. Cytomixis was shown to be more common for intratissular interactions, and cytomixis in the tapetum exhibited taxon-specific features, both structural and temporal. Nuclear migration in the microsporocytes mostly occurred during the zygotene–pachytene and exhibited certain synchrony with cytomixis in the tapetum. Intertissular cytomictic interactions (between the tapetum and the microsporocytes) were detected only in monocotyledonous plant anthers. Intertissular interactions may reflect more intense competition for space between the tapetum and the microsporocytes during the differentiation of anther tissues. The polyploid nuclei of the tapetum and the syncytia are powerful acceptors that can compete with the microsporocytes and attract the chromatin during translocation of the latter. The absence of intertissular interactions in dicotyledonous plants may be indicative of a better balance between the processes of differentiation of somatic and generative tissues of the microsporangium as compared to monocotyledonous plants.     

Comparison of the interaction of mono- and oligovalent ligands with cholera toxin. Demonstration of aggregate formation at low ligand concentrations.

The stimulation by cholera toxin of adenylate cyclase in Chinese hamster ovarian cells could be inhibited by various ligands. The latter have been shown to contain the structural oligosaccharide entities required for binding to cholera toxin, established as Galbeta1 leads to 3GalNAcbeta1 leads to 4Gal3 comes from 2alphaNeuAc. The different inhibitory potency of the ligands thereby correlates with the size of the aggregates formed with the toxin, which in turn depends on the valency of the ligands. The conclusion is drawn from a comparison of the interaction of cholera toxin and its B-protomer with ganglioside II3NeuAc-GgOse4-Cer, the newly synthesized bis-(monosialo-gangliotetraityl)amine and monosialogangliotetraose. In a double diffusion test cholera toxin B-protomer precipitated with the ganglioside II3 NeuAcGgOSE4-Cer and the divalent ligand bis(monosialo-gangliotetraityl)amine, suggesting the formation of high molecular weight aggregates, whereas no precipitation was observed with the monovalent monosialo-gangliotetraose. By ultracentrifugation analysis, aggregate formation of the cholera toxin B-protomer could be demonstrated with the ganglioside II3 NeuAc-GgOse4-Cer and bis(monosialo-gangliotetraityl)amine at a concentration at which the ganglioside was assumed to be monodisperse. Ganglioside/cholera toxin B-protomer complexes sediment faster than those of the toxin and bis(monosialo-gangliotetraityl)amine, suggesting higher aggregation of cholera toxin B-protomer with the former. On the other hand, no sedimentation with monosialo-gangliotetraose was observed. By equilibrium displacement dialysis, however, a comparable high affinity of binding to cholera toxin B-protomer of both the mono- and divalent oligosaccharides was demonstrated. Furthermore, values for the maximal concentration of the bound ligand from these binding experiments with cholera toxin B-protomer established molar ratios of ligand to protein of 4 to 1 and 2 to 1 for monosialo-gangliotetraose and bis(monosialo-gangliotetraityl)amine, respectively. From the results it is concluded that the lipophilic moiety of the ganglioside is not directly involved in the binding process to the toxin protein but leads to an oligovalency of this ligand, due to formation of micellar or submicellar structures.     

A novel, specific interaction involving the Csk SH3 domain and its natural ligand.

C-terminal Src kinase (Csk) takes part in a highly specific, high affinity interaction via its Src homology 3 (SH3) domain with the proline-enriched tyrosine phosphatase PEP in hematopoietic cells. The solution structure of the Csk-SH3 domain in complex with a 25-residue peptide from the Pro/Glu/Ser/Thr-rich (PEST) domain of PEP reveals the basis for this specific peptide recognition motif involving an SH3 domain. Three residues, Ala 40, Thr 42 and Lys 43, in the SH3 domain of Csk specifically recognize two hydrophobic residues, Ile 625 and Val 626, in the proline-rich sequence of the PEST domain of PEP. These two residues are C-terminal to the conventional proline-rich SH3 domain recognition sequence of PEP. This interaction is required in addition to the classic polyproline helix (PPII) recognition by the Csk-SH3 domain for the association between Csk and PEP in vivo. NMR relaxation analysis suggests that Csk-SH3 has different dynamic properties in the various subsites important for peptide recognition.     

Identification and characterization of ligands for L-selectin in the kidney. II. Expression of chondroitin sulfate and heparan sulfate proteoglycans reactive with L-selectin

Ligands for the leukocyte adhesion molecule L-selectin are expressed not only in lymph node high endothelial venules (HEV) but also in the renal distal tubuli. Here we report that L-selectin-reactive molecules in the kidney are chondroitin sulfate and heparan sulfate proteoglycans of 500-1000 kDa, unlike those in HEV bearing sialyl Lewis X-like carbohydrates. Binding of L-selectin to these molecules was mediated by the lectin domain of L-selectin and required divalent cations. Binding was inhibited by chondroitinase and/or heparitinase but not sialidase. Thus, L-selectin can recognize chondroitin sulfate and heparan sulfate glycosaminoglycans structurally distinct from sialyl Lewis X-like carbohydrates.     

Leukocyte entry into sites of inflammation requires overlapping interactions between the L-selectin and ICAM-1 pathways.

Leukocyte interactions with vascular endothelium during inflammation depend on cascades of adhesion molecule engagement, particularly during selectin-mediated leukocyte rolling. Leukocyte rolling is also facilitated by members of the integrin and Ig families. Specifically, leukocyte rolling velocities during inflammation are significantly increased in ICAM-1-deficient mice, with ICAM-1 expression required for optimal P- and L-selectin-mediated rolling. Elimination of ICAM-1 expression in L-selectin-deficient mice significantly reduces leukocyte rolling. Whether disrupted leukocyte rolling in L-selectin and ICAM-1 double-deficient (L-selectin/ICAM-1-/-) mice affects leukocyte entry into sites of inflammation in vivo was assessed in the current study by using experimental models of inflammation; thioglycollate-induced peritonitis, chemokine-induced neutrophil migration to the skin, delayed-type hypersensitivity responses, rejection of allogeneic skin grafts, and septic shock. In many cases, the loss of both L-selectin and ICAM-1 expression dramatically reduced leukocyte migration into sites of inflammation beyond what was observed with loss of either receptor alone. In fact, the effects from loss of both L-selectin and ICAM-1 effectively eliminated multiple chronic inflammatory responses in L-selectin/ICAM-1-/- mice. By contrast, the combined loss of L-selectin and ICAM-1 expression had minimal effects on the generation of Ag-specific T cell responses or humoral immunity. Thus, members of the selectin and Ig families function synergistically to mediate optimal leukocyte rolling and entry into tissues, which is essential for the generation of effective inflammatory responses in vivo.     

P- and E-selectins recognize sialyl 6-sulfo Lewis X, the recently identified L-selectin ligand

Recently we identified sialyl 6-sulfo Le(x) as a major L-selectin ligand on high endothelial venules of human peripheral lymph nodes. In this study we investigated the ligand activity of sialyl 6-sulfo Le(x) to E- and P-selectins and compared it with the binding activity of conventional sialyl Le(x), by using cultured human lymphoid cells expressing both carbohydrate determinants. The results of the recombinant selectin binding studies and the nonstatic monolayer cell adhesion assays indicated that both sialyl 6-sulfo Le(x) and conventional sialyl Le(x) served as ligand for E- and P-selectins, while L-selectin was quite specific to sialyl 6-sulfo Le(x). Anti-PSGL-1 antibodies as well as O-sialoglycoprotein endopeptidase treatment almost completely abrogated the binding of P-selectin but barely affected the binding of E-selectin, indicating that these carbohydrate determinants carried by O-glycans of PSGL-1 selectively serves as a ligand for P-selectin, while the ligand for E-selectin is not restricted to PSGL-1 nor to O-sialoglycoprotein endopeptidase-sensitive glycans. The binding of L-selectin was markedly reduced by O-sialoglycoprotein endopeptidase treatment but only minimally affected by anti-PSGL-1 antibodies, indicating that O-glycans carrying sialyl 6-sulfo Le(x) were the major L-selectin ligands, while PSGL-1 was only a minor core protein for L-selectin in these cells. These results indicated that each member of the selectin family has a distinct ligand binding specificity.     

Comparison of L-selectin and E-selectin ligand specificities: the L-selectin can bind the E-selectin ligands sialyl Le(x) and sialyl Le(a).

The L- and E-selectins are leukocyte and endothelial cell surface molecules which mediate leukocyte-endothelial cell adhesion by interacting with carbohydrate ligands. In the present study we find that L-selectin, like E-selectin, can interact with synthetic neoglycoproteins containing Sialyl Le(x) (Neu5Ac alpha 2-3Gal beta 1-4[Fuc alpha 1-3]GlcNAc beta-R), or Sialyl Le(a) (Neu5Ac-alpha 2-3Gal beta 1-3[Fuc alpha 1-4]GlcNAc beta-R). Additionally, both the E-selectin and L-selectin can bind the peripheral lymph node addressin, a high endothelial venule ligand for L-selectin. Despite overlapping interactions, the L- and E-selectins discriminate between their native ligands. The peripheral lymph node addressin is a preferential ligand for L-selectin; and furthermore, L-selectin expressing cells do not interact detectably with the cutaneous lymphocyte antigen, a native glycoprotein ligand for E-selectin found on a subset of lymphocytes associated with the skin.     

Ionic ligand interactions with the intracellular loop of the sodium-calcium exchanger. Modulation by ATP

In the last decade, there has been a large increase in the study of the Na(+)/Ca(2+) exchanger due to its implications in physiological and pathophysiological processes at the cell and organ levels. Key areas of these studies have been molecular biology, regulation and physiology-pathophysiology of the exchanger. There are three main types of regulation that take place at the large intracellular loop of the Na(+)/Ca(2+) exchanger: (i) ionic (sodium inactivation, calcium regulation and proton inhibition), (ii) metabolic (ATP as phosphoryl group donor), and (iii) genetic (alternative splicing). This review analyzes the most recent data on the mutual interactions of regulatory ionic ligands (Ca(2+), Na(+), H(+)) and how they are secondarily modulated by MgATP, emphasizing the importance of the binding of Ca(2+) to its regulatory site as an essential requirement for the exchange function. Intracellular protons and sodium inhibit the Na(+)/Ca(2+) exchanger by reducing the apparent affinity of the Ca(i)-regulatory site for Ca(2+). Although the metabolic pathways are different in the mammalian heart (membrane lipids) and squid nerve cells (soluble cytosolic regulatory protein), the final mechanism for the protective effect of MgATP is the same: a reduction of Na(i)(+)-H(i)(+) binding affinities facilitating the attachment of Ca(2+) to its regulatory site. Kinetic models, which partially analyzed some of these ionic and metabolic interactions, can be integrated into a single scheme where the Ca(i)-regulatory site plays a central role.     

3H]Para-amino-clonidine: a novel ligand which binds with high affinity to alpha-adrenergic receptors.

Para-amino-clonidine (PAC) is an α-adrenergic agonist with extraordinarily high potency in some peripheral tissues. We have demonstrated the labeling of α-adrenergic binding sites in central and peripheral tissues with [³H]PAC and compared properties of this binding to those of [³H]clonidine. [³H]PAC binds saturably with a dissociation constant (K_D) of about 0.9 nM to rat cerebral cortex membranes. It has about 2–3 times the affinity of [³H]clonidine for α-receptor binding sites. The greater affinity is attributable mainly to a slower dissociation of [³H]PAC than [³H]clonidine from binding sites. The relative and absolute potencies of various adrenergic agonists and antagonists in competing for [³H]PAC and [³H]clonidine binding are essentially the same. [³H]PAC can also be utilized to label α-adrenergic binding sites in the kidney and spleen where the relative potencies of PAC and clonidine are the same as in the brain.