A modified risk assessment to establish molybdenum standards for land application of biosolids.

The USEPA standards (40 CFR Part 503) for the use or disposal of sewage sludge (biosolids) derived risk-based numerical values for Mo for the biosolids --> land --> plant --> animal pathway (Pathway 6). Following legal challenge, most Mo numerical standards were withdrawn, pending additional field-generated data using modern biosolids (Mo concentrations <75 mg kg(-1) and a reassessment of this pathway. This paper presents a reevaluation of biosolids Mo data, refinement of the risk assessment algorithms, and a reassessment of Mo-induced hypocuprosis from land application of biosolids. Forage Mo uptake coefficients (UC) are derived from field studies, many of which used modern biosolids applied to numerous soil types, with varying soil pH values, and supporting various crops. Typical cattle diet scenarios are used to calculate a diet-weighted UC value that realistically represents forage Mo exposure to cattle. Recent biosolids use data are employed to estimate the fraction of animal forage (FC) likely to be affected by biosolids applications nationally. Field data are used to estimate long-term Mo leaching and a leaching correction factor (LC) is used to adjust cumulative biosolids application limits. The modified UC and new FC and LC factors are used in a new algorithm to calculate biosolids Mo Pathway 6 risk. The resulting numerical standards for Mo are cumulative limit (RPc)=40 kg Mo ha(-1), and alternate pollutant limit (APL) = 40 mg Mo kg(-1) We regard the modifications to algorithms and parameters and calculations as conservative, and believe that the risk of Mo-induced hypocuprosis from biosolids Mo is small. Providing adequate Cu mineral supplements, standard procedure in proper herd management, would augment the conservatism of the new risk assessment.

The discovery of sulfonylated dipeptides as potent VLA-4 antagonists.

Directed screening of a carboxylic acid-containing combinatorial library led to the discovery of potent inhibitors of the integrin VLA-4. Subsequent optimization by solid-phase synthesis afforded a series of sulfonylated dipeptide inhibitors with structural components that when combined in a single hybrid molecule gave a sub-nanomolar inhibitor as a lead for medicinal chemistry. Preliminary metabolic studies led to the discovery of substituted biphenyl derivatives with low picomolar activities. SAR and pharmacokinetic characterization of this series are presented.

Arginine 206 of the C5a receptor is critical for ligand recognition and receptor activation by C-terminal hexapeptide analogs.

C5a is a 74-amino-acid glycoprotein whose receptor is a member of the rhodopsin superfamily. While antagonists have been generated to many of these receptors, similar efforts directed at family members whose natural ligands are proteins have met with little success. The recent development of hexapeptide analogs of C5a has allowed us to begin elucidation of the molecular events that lead to activation by combining a structure/activity study of the ligand with receptor mutagenesis. Removal of the hexapeptide's C-terminal arginine reduces affinity by 100-fold and eliminates the ability of the ligand to activate the receptor. Both the guanidino side chain and the free carboxyl of the arginine participate in the interaction. The guanidino group makes the energy-yielding contact with the receptor, while the free carboxylate negates "electrostatic" interference with Arg-206 of the receptor. It is the apparent movement Arg-206 induced by this set of interactions that is responsible for activation, since conversion of Arg-206 to alanine eliminates the agonist activity of the hexapeptides. Surprisingly, activation is a nearly energy-neutral event and may reflect the binding process rather than the final resting site of the ligand.

Development of C5a receptor antagonists. Differential loss of functional responses.

C5a is a 74-amino acid glycoprotein generated on activation of the C system. The responses evoked by C5a, both in vitro and in vivo, and its association with inflammatory diseases, suggest that a receptor antagonist would be of considerable therapeutic importance. However, efforts at generating antagonists have so far been unsuccessful. Structure/activity studies of the C terminus of C5a have generated peptide analogues with nanomolar affinities, but all of these retain strong agonist properties. We now report hexapeptides of the form NMePhe-Lys-Pro-dCha-X-dArg in which increasing aromaticity at position 5 leads to a progressive loss of agonism with little change in binding affinity. The different responses induced by C5a are lost in the order: degranulation before Ca(2+)-flux before chemotaxis. We also describe the first full antagonist of C5a, because the peptide in which x = Trp is not only devoid of all agonist properties, but it inhibits C5a induced degranulation and C5a stimulated G protein activation.

The amino terminus of the human C5a receptor is required for high affinity C5a binding and for receptor activation by C5a but not C5a analogs.

The binding domain of the human C5a receptor consists of two distinct and physically separable subsites. One of these sites binds the C-terminal 8 amino acids of C5a and is as yet undefined, while the second site lies in the N terminus of the receptor and interacts with the core of C5a. Two deletion mutants were prepared to probe the importance of this second site. Removal of residues 2-22 decreased the binding affinity for C5a by 600-fold, while extending the deletion through residue 30 caused a further 75-fold decrease. Thus, the N terminus is responsible for at least 45% of the total energy for the binding of C5a. The five aspartic acids present in the deleted segments appear to be critical residues, as their conversion to alanines accounts for most of the affinity lost in the two truncations. Despite its importance for binding, the N terminus is not necessary for signal transduction, as a C-terminal peptide analog of C5a was able to stimulate G protein activation and to generate a Ca2+ flux through a receptor lacking residues 2-22. However, intact C5a was a very poor activator of this truncated receptor. These results imply that interaction between the N terminus of the receptor and C5a produces a conformational change in C5a that allows it's C terminus to properly interact with and activate the receptor.

Induction, characterization, and functional coupling of the high affinity chemokine receptor for RANTES and macrophage inflammatory protein-1 alpha upon differentiation of an eosinophilic HL-60 cell line.

Eosinophilic differentiation of a pro-eosinophilic HL-60 cell line resulted in the induction of a high affinity RANTES/macrophage inflammatory protein-1 alpha receptor. The induced receptor is biochemically indistinguishable in RANTES equilibrium-binding studies from the monocytic receptor expressed on THP-1 cell membranes. Continued expression of the receptor requires the continuous presence of the inducing stimulus, and receptor site number declines without a loss of binding affinity with a t1/2 of 11.5 h on withdrawal of the inducing stimulus. The induced receptor is capable of three physiologic measures of receptor coupling, namely, ligand-induced Ca2+ fluxes, priming of the respiratory burst, and chemotaxis. Dose-dependent Ca2+ fluxes were elicited upon increasing concentrations of RANTES and MIP-1 alpha whereas no response was measured upon addition of MIP-1 beta or MCP-1. In addition, desensitization studies demonstrated that previous exposure to either RANTES or MIP-1 alpha almost completely inhibits a Ca2+ flux upon subsequent exposure to either ligand. Priming of the respiratory burst to PMA in differentiated cells by human rRANTES was more effective than priming by IL-5 or granulocyte-macrophage-CSF, whereas undifferentiated cells failed to secrete superoxide anion. In addition, differentiated cells underwent chemotaxis in response to RANTES. This provides the first evidence for the induction of a C-C chemokine receptor upon eosinophilic differentiation of a leukocyte cell line, and is in keeping with the demonstrated ability of human RANTES to induce the rapid formation of eosinophilic inflammatory sites.

Two-site binding of C5a by its receptor: an alternative binding paradigm for G protein-coupled receptors.

The guanine nucleotide-binding protein-coupled receptor superfamily binds a vast array of biological messengers including lipids, odorants, catecholamines, peptides, and proteins. While some small molecules bind to these receptors at a single interhelical site, we find that the binding domain on the receptor for the inflammatory protein C5a is more complex and consists of two distinct subsites. This more elaborate motif appears to be an evolutionary adaptation of the simpler paradigm to which a second interaction site has been added in the receptor N terminus. Surprisingly, occupation of only one of the subsites is required for receptor activation. The two-site motif is not unique to the C5a receptor but appears to be widely used by the superfamily to accommodate macromolecular ligands.

Formation of eosinophilic and monocytic intradermal inflammatory sites in the dog by injection of human RANTES but not human monocyte chemoattractant protein 1, human macrophage inflammatory protein 1 alpha, or human interleukin 8.

Equilibrium binding studies on canine mononuclear and granulocytic cells allow the identification of a single high affinity receptor for the human C-C chemokine RANTES (dissociation constant, 14 +/- 8 pM), that, in contrast to the human RANTES receptor, has no affinity for human macrophage inflammatory protein 1 alpha (hMIP-1 alpha). A single intradermal injection of hRANTES in dog resulted in eosinophil- and macrophage-rich inflammatory sites within 4 h. Cell infiltration peaked at 16-24 h after hRANTES injection. There was histological evidence of intravascular activation of eosinophils at 4 h, although eosinophils in the vasculature and interstitium contained apparently intact granules. Monocytes were the predominant cells adherent to venular endothelium at 16-24 h. Human MIP-1 alpha elicited no response in canine dermis, whereas monocyte chemoattractant protein 1 caused mild perivascular cuffing with monocytes. In contrast, human interleukin 8 induced a neutrophilic dermal infiltrate that was maximal by 4 h after challenge. This provides the first direct evidence in vivo that RANTES has significant proinflammatory activity and, in addition, could be a mediator in atopic pathologies characterized by eosinophilic and monocytic inflammatory responses.

Characterization and species distribution of high affinity GTP-coupled receptors for human rantes and monocyte chemoattractant protein 1.

Equilibrium binding studies with recombinant human chemoattractant cytokines Rantes and monocyte chemoattractant protein 1 (MCP-1) on monocytic THP-1 cells have allowed the functional identification of two distinct receptors for C-C chemokines. One is a novel oligospecific receptor with high affinity for Rantes (50% maximal inhibitory concentration [IC50], 0.68 nM) and low affinity (IC50, 35 nM) for MCP-1, while the other is the previously described specific receptor for MCP-1 (IC50, 0.5 nM). Receptor affinity for Rantes is enhanced on preparation of isolated membranes with a 12-fold decrease in receptor Kd. The basis of this enhancement is not understood. The Rantes receptor appears to be G protein linked, as binding activity is abolished by guanosine 5'-O-(3-thiotriphosphate) (IC50, 7.3 nM). In contrast to the consequences of MCP-1 binding, we were unable to demonstrate ligand-dependent calcium fluxes on binding of Rantes to human monocytes or THP-1 cells. The binding of Rantes and MCP-1 to mononuclear cells from dog, rabbit, and rat were tested. While high affinity binding could be demonstrated in dog and rabbit, differences in ligand-induced Ca2+ fluxes could be shown between species. This suggests that receptor-ligand interactions and receptor coupling is best examined with autologous receptors and cytokine.

Construction and phosphorylation of a fusion protein Hu-IFN-alpha A/gamma.

A protein consisting of human (Hu)-IFN-alpha A to which the COOH-terminal 16 amino acids of Hu-IFN-gamma were fused was prepared by constructing an expression vector by oligonucleotide-directed mutagenesis. The hybrid protein Hu-IFN-alpha A/gamma was expressed under the control of phage lambda PL promoter. The protein was purified with the use of a monoclonal antibody against Hu-IFN-alpha or the COOH-terminal amino acid sequence of Hu-IFN-gamma. The purified protein exhibited a single major band on sodium dodecyl sulfate-polyacrylamide gel electrophoresis and has antiviral activity on human and bovine cells. Unlike Hu-IFN-alpha A, but similar to Hu-IFN-gamma, the hybrid Hu-IFN-alpha A/gamma can be phosphorylated by [gamma 32P]ATP and cAMP-dependent protein kinase. The phosphorylated molecule binds to the IFN-alpha/beta receptor. The introduction of a phosphorylation site into Hu-IFN-alpha A by fusion of the region of Hu-IFN-gamma which contains the phosphorylation site provides a new reagent for studies of receptor binding, pharmacokinetics, and other studies where labeled interferons are useful. Furthermore, the introduction of phosphorylation sites into proteins provides a new principle for the preparation of a wide variety of reagents for many purposes.