Modification of a receptor-binding surface of epidermal growth factor (EGF): analogs with enhanced receptor affinity at low pH or at neutrality.

Six mutants of human epidermal growth factor (EGF), which carry single point substitutions within a surface patch proposed to juxtapose the bound receptor, were prepared and characterized for receptor affinity and mitogenicity. Receptor affinities relative to EGF are G12Q > H16D > Y13W > Q43A approximately = H16A approximately = EGF >> L15A. Notably, the reduced receptor affinity of mutant L15A indicates that Leu15 probably contributes substantially to receptor binding whereas unaltered receptor affinities observed for analogs H16A and Q43A indicate that neither His16 nor Gln43 contributes significantly to this interaction.

Synthesis and characterization of a monomeric mutant Cu/Zn superoxide dismutase with partially reconstituted enzymic activity.

A monomeric analog of human Cu/Zn superoxide dismutase (F50E/G51E SOD), previously characterized and found to have reduced enzymic activity, was here further modified by replacing Glu133 with Gln. This substitution does not dramatically affect the coordination geometry at the active site, but enhances enzymic activity, and also increases the affinity for anions at the active site. This behavior parallels earlier published results in which this point mutation was made in the dimeric wild-type enzyme.

Heterologous expression of selenium-dependent glutathione peroxidase affords cellular resistance to paraquat.

Transfection of murine NIH3T3 fibroblasts and human MCF7 breast carcinoma cells with a pSV2-derived eukaryotic expression vector for human cytosolic glutathione peroxidase resulted in clones with increased glutathione peroxidase activity. This heterologous expression indicates that murine cells recognize the human "selenocysteine insertion sequence" in the 3' untranslated region of the mRNA which facilitates insertion of selenocysteine directed by the opal codon. Though most clones from both cell lines eventually lost their enhanced glutathione peroxidase activity despite continuous selection on G418, some NIH3T3 clones retained enhanced enzyme activity without continuous G418 exposure.

IL-8 induces neutrophil chemotaxis predominantly via type I IL-8 receptors.

IL-8 is a potent proinflammatory cytokine that has a key role in the recruitment and activation of neutrophils during inflammation. IL-8 reacts with neutrophils via two distinct types of IL-8-R. Receptor-specific Abs were raised against peptides derived from the first extracellular domain of each IL-8-R.

High yields of interleukin-8 produced by a synthetic gene expressed in Escherichia coli and purified with a single antibody affinity column.

We developed a highly efficient expression system for the production of interleukin-8 (IL-8) in Escherichia coli. A synthetic gene used in the vector was designed to code for the 72-amino-acid form of IL-8 and incorporate additional new restriction sites. IL-8 was expressed in very large amounts in the periplasmic space and extracted by a gentle method which did not utilize denaturants.