Daniel J. O'Shannessy

Cloning and functional characterization of a novel human CC chemokine that binds to the CCR3 receptor and activates human eosinophils.

Eotaxin has been found to bind exclusively to a single chemokine receptor, CCR3. Using expression sequence tag screening of an activated monocyte library, a second chemokine has been identified; it was expressed and purified from a Drosophila cell culture system and appears to only activate CCR3. Eotaxin‐2, MPIF‐2, or CKβ‐6, is a human CC chemokine with low amino acid sequence identity to other chemo‐ kines. Eotaxin‐2 promotes chemotaxis and Ca2+ mobilization in human eosinophils but not in neutrophils or monocytes. Cross‐desensitization calcium mobilization experiments using purified eosinophils indicate that eotaxin and MCP‐4, but not RANTES, MIP‐lα, or MCP‐3, can completely cross‐desensitize the calcium response to eotaxin‐2 on these cells, indicating that eotaxin‐2 shares the same receptor used by eotaxin and MCP‐4. Eotaxin‐2 was the most potent eosinophil chemoattractant of all the chemokines tested. Eotaxin‐2 also displaced 125I‐eotaxin bound to the cloned CCR3 stably expressed in CHO cells (CHO‐CCR3) and to freshly isolated human eosinophils with affinities similar to eotaxin and MCP‐4. l25I‐Eotaxin‐2 binds with high affinity to eosinophils and both eotaxin and cold eotaxin‐2 displace the ligand with equal affinity. Eotaxin and eotaxin‐2 promote a Ca2+ transient in RBL‐2H3 cells stably transfected with CCR3 (RBL‐2H3‐CCR3) and both ligands cross‐desensitized the response of the other but not the response to LTD4. The data indicate that eotaxin‐2 is a potent eosinophil chemotactic chemokine exerting its activity solely through the CCR3 receptor. J. Leukoc. Biol. 62: 667–675; 1997.

NOVEL, SELECTIVE MECHANISM-BASED INHIBITORS OF THE HERPES PROTEASES

Abstract A novel class of inhibitor of the herpes proteases acting upon the catalytic apparatus by forming covalent complexes are described. Two new families of inhibitor, the spirocyclopropyl oxazolones and the benzylidine N-sulphonyloxyimidazolones, have been shown to be submicromolar inhibitors of HSV-2 and HCMV proteases which are selective relative to a panel of standard serine proteases.

Antibodies biotinylated via sugar moieties.

Publisher Summary This chapter describes a number of hydrazide derivatives of biotin including biotin hydrazide (BHZ), biotin aminocaproylhydrazide (BACH), and biocytin hydrazide. Hydrazides are very efficient nucleophiles for condensation with aldehydes. Therefore, the reaction scheme involves the generation of aldehydes specifically on the oligosaccharide moieties of antibodies. While the oxidation reaction is proceeding, a Pharmacia PD-10 gel filtration column is equilibrated with the same acetate–sodium chloride buffer used to prepare the antibody. Immediately after the oxidation is complete, the oxidized antibody solution is applied to the PD-10 column and subsequently eluted with 3.5 ml of the same buffer. The vast array and ready availability of avidin and streptavidin conjugates make biotinylation of antibodies amenable to any detection, quantification, or purification system currently in use employing avidin–biotin technology. Specific biotinylation of sugar moieties may be used to assess the purity of an antibody preparation with respect to glycoprotein contamination and to analyze the oligosaccharide distribution of the antibody.

Measurement of protein interaction bioenergetics: Application to structural variants of anti-sCD4 antibody

This chapter has described a bioenergetic analysis of the interaction of sCD4 with an IgG1 and two IgG4 derivatives of an anti-sCD4 MAb. The MAbs have identical VH and VL domains but differ markedly in their CH and CL domains, raising the question of whether their antigen-binding chemistries are altered. We find the sCD4-binding kinetics and thermodynamics of the MAbs are indistinguishable, which indicates rigorously that the molecular details of the binding interactions are the same. We also showed the importance of using multiple biophysical methods to define the binding model before the bioenergetics can be appropriately interpreted. Analysis of the binding thermodynamics and kinetics suggests conformational changes that might be coupled to sCD4 binding by these MAbs are small or absent.

Biological and biophysical characterization of recombinant soluble human E-selectin purified at large scale by reversed-phase high-performance liquid chromatography

A first step in the development of a high-throughput screening assay for antagonists of human E-selectin is the purification and characterization of the selectin. In the present paper we describe a single-step, rapid, reversed-phase HPLC purification protocol for the recombinant, soluble form of human E-selectin (rshE-selectin) produced in Chinese hamster ovary cells. The procedure resulted in high protein yields with recoveries of greater than 98%. Characterization of the reversed-phase purified rshE-selectin showed this product to be analogous to rshE-selectin purified using conventional chromatographic techniques with respect to biological activity and molecular shape. However, the carbohydrate composition of reversed-phase purified rshE-selectin, which had been variable with conventionally purified material, was found to be constant across several isolations. The protocol described herein eliminated the high mannose component associated with previously purified rshE-selectin and provided a uniform carbohydrate composition for additional experimental studies, such as NMR. This fact, coupled with the high yield and simplicity of the present purification scheme are distinct advantages over those previously published. It is expected that other mammalian selectins, such as P-selectin and L-selectin, would also be amenable to reversed-phase HPLC purification.