In a recent study, we demonstrated that glycosaminoglycan (GAG) binding and oligomerization are essential for the in vivo function of the chemokines MCP-1/ CCL2, RANTES/CCL5, and MIP-1␤/CCL4 (1). Binding to the GAG chains of cell surface proteoglycans is thought to facilitate the formation of high localized concentrations of chemokines, which in turn provide directional signals for leukocyte migration. To understand the molecular details of the chemokine-GAG interaction, in the present study we identified the GAG binding epitopes of MCP-1/CCL2 by characterizing a panel of surface alanine mutants in a series of heparin-binding assays. Using sedimentation equilibrium and cross-linking methods, we also observed that addition of heparin octasaccharide induces tetramer formation of MCP-1/CCL2. Although MCP-1/CCL2 forms a dimer in solution, both a dimer and tetramer have been observed by x-ray crystallography, providing a glimpse of the putative heparin-bound state. When the GAG binding residues are mapped onto the surface of the tetramer, the pattern that emerges is a continuous ring of basic residues encircling the tetramer, creating a positively charged surface well suited for binding GAGs. The structure also suggests several possible functional roles for GAGinduced oligomerization beyond retention of chemokines at the site of production.

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Chemokines orchestrate the migration of leukocytes in the context of homeostasis and inflammation. In addition to interactions of chemokines with receptors on migrating cells, these processes require interactions of chemokines with glycosaminoglycans (GAGs) for cell surface localization. Most chemokines are basic proteins with Arg/Lys/His residue clusters functioning as recognition epitopes for GAGs. In this study we characterized the GAG-binding epitopes of the chemokine I-TAC/CXCL11. Four separate clusters of basic residues were mutated to alanine and tested for their ability to bind to GAGs in vitro and to activate the receptor, CXCR3. Mutation of a set of basic residues in the C-terminal helix (the 50s cluster, 57 KSKQAR 62 ) along with Lys 17 , significantly impaired heparin binding in vitro, identifying these residues as components of the dominant epitope. However, this GAG mutant retained nearly wild type receptor binding affinity, and its ability to induce cell migration in vitro was only mildly perturbed. Nevertheless, the mutant was unable to induce cell migration in vivo, establishing a requirement of CXCL11 for GAG binding for in vivo function. These studies also led to some interesting findings. First, CXCL11 exhibits conformational heterogeneity, as evidenced by the doubling of peaks in its HSQC spectra. Second, it exhibits more than one affinity state for both heparin and CXCR3, which may be related to its structural plasticity. Finally, although the binding affinities of chemokines for GAGs are typically weaker than interactions with receptors, the high affinity GAG binding state of CXCL11 is comparable with typical receptor binding affinities, suggesting some unique properties of this chemokine.Chemokines belong to a family of small chemotactic cytokines that selectively recruit and activate specific leukocytes during inflammation and routine immunosurveillance (1, 2).The chemokines of all four subclasses (CC, CXC, CX3C, and C) have a remarkably conserved three-dimensional tertiary structure, but many form dimers, tetramers, and higher order oligomers, and although monomeric forms are sufficient for cell migration in vitro (3-5), for some chemokines, oligomerization is required for function in vivo (6, 7). All chemokines exert their biological activity by binding to seven-transmembrane G protein-coupled receptors, which are also subdivided into four classes analogous to the ligand classification (8). Additionally, many chemokines interact with the glycosaminoglycan (GAG) 3 moieties of proteoglycans on endothelial cells and the extracellular matrix (6). GAGs enable the surface immobilization of chemokines, thereby creating haptotactic gradients in order to direct leukocytes to sites of inflammation (9). As demonstrated with a series of chemokine mutants that were impaired in their ability to bind GAGs, when the GAG interaction is disrupted, chemokines lose the ability to efficiently recruit cells in vivo, even when chemotaxis in vitro is unperturbed (7).GAGs are negatively charged linear polysacc...

Summary Effects of the herbicide EL‐107 (N‐[3‐(1‐ethyl‐1‐methylpropyl)‐5‐isoxazolyl]‐2,6‐dimethoxy‐benzamide) on the growth of dicotyledonous plants are described. The herbicide did not inhibit germination but reduced the growth of rape (Brassica napus L.) by half at 0.0057 mg l−1. The most characteristic symptom induced was a swelling of the apical regions, and histological observations of root tips of Polygonum persicaria and rape revealed a progressve disappearance of the meristematic zone, which was replaced by enlarged cells almost devoid of cytoplasm. Growth of cells of Acer pseudoplatanus L. and soybean (Glycine max L.) cultured in suspension was also inhibited by EL‐107, which induced a characteristic change in the cell morphology to an enlarged, rounded shape. EL‐107 apparently did not block cell division; at a high concentration it produced some inhibition of protein synthesis, but in Acer cells only. In both types of cell cultures it inhibited, to some extent, the incorporation of glucose into cell wall material.

Galium aparine plants were treated at the two‐whorl stage with technical‐grade phenmedipham. The active ingredient was applied at 10 doses as an acetone:water solution, without emulsifier, alone or in mixture with an ester of oleic acid (methyl, butyl, octyl, dodecanyl, octadecanyl). The plant response was described by a log‐logistic regression model. Phenmedipham applied alone did not kill the plants even at the highest dose (1621 g a.i. ha−1). In contrast, addition of any alkyl oleate to phenmedipham killed the plants at the 200–400 g a.i. ha−1 doses. The alkyl oleates differently affected activity of phenmedipham. Their effects could be ranked as follows: butyl ≥ methyl; octyl> methyl; dodecanyl ≤ methyl; octadecanyl < methyl oleate. Thus, butyl and octyl oleate promoted phenmedipham activity more than methyl oleate.