The 29 kDa protein from the larval epidermis of the tobacco hornworm, Manduca sexta, that specifically bound photoaffinity analogs of JH I and JH II was produced by a recombinant baculovirus (rJP29). The higher of the two molecular weight forms made corresponded to a protein that could be formed by read‐through of the TGA termination codon to the following TAA. The previously reported, apparent high affinity binding of [methyl‐3H]‐JH I by rJP29 as measured by the dextran‐coated charcoal (DCC) assay [Palli et al., Proc Natl Acad Sci USA 91:6191–6195 (1994)] was found to be artifactual due to endogenous cellular esterases that co‐purified with rJP29 through both DEAE cellulose and MonoQ chromatography. These esterases converted the 10–20 nM labelled JH to JH I acid and [3H]‐methanol during the 1 h incubation at room temperature. Additionally, DEAE fractions containing rJP29 or from wild‐type virus‐infected cells were found to bind nonspecifically high amounts of 12, 13‐3H]‐JH I acid in the DCC assay. Neither rJP29 nor the cellular esterases had JH esterase activity when assayed on a series of thioether surrogate substrates. When separated from these contaminating esterases either by hydroxylapatite or affinity chromatography, rJP29 showed little or no detectable binding of [12,13‐3H]‐JH I. Yet purified rJP29 bound EBDA, the photoaffinity analog of JH I; and this binding was prevented by retinol, JH I, methyl farnesoate, methoprene, and xanthophyll, but not by farnesol and 20‐hydroxyecdysone. Therefore, JP29 is not a high affinity JH receptor. © 1996 Wiley‐Liss, Inc.
A diverse group of proteins has evolved to bind and metabolize insect juvenile hormones (JHs). Synthetic radiolabeled JHs and their photoaffinity analogs have enabled us to isolate and characterize JH binding proteins (JHBPs), a putative nuclear JH receptor, JH esterases (JHEs), JH epoxide hydrolases (JHEHs), and methyl farnesoate binding proteins (MFBPs). Highlights of recent progress on structural characterization of JHBPs and JHEHs of two lepidopterans will be described. Efforts to identify MFBPs of penaeid shrimp will be discussed, and the discovery of a possible vertebrate JHBP will be presented.
A diverse group of proteins has evolved to bind and metabolize insect juvenile hormones (JHs). Synthetic radiolabeled JHs and their photoaffinity analogs have enabled us to isolate and characterize JH binding proteins (JHBPs), a putative nuclear JH receptor, JH esterases (JHEs), JH epoxide hydrolases (JHEHs), and methyl farnesoate binding proteins (MFBPs). Highlights of recent progress on structural characterization of JHBPs and JHEHs of two lepidopterans will be described. Efforts to identify MFBPs of penaeid shrimp will be discussed, and the discovery of a possible vertebrate JHBP will be presented. © 1996 Wiley‐Liss, Inc.
Macrophage adhesion to a wide variety of substrates has been measured, but no systematic study of the influence of specific substrate chemical properties on adhesion is available. These studies were conducted using two series of materials, copolymers of hydroxyethyl methacrylate (HEMA) and ethyl methacrylate (EMA) and copolymers of hydroxystyrene and styrene, to determine the effect of a single chemical property, polar character, on adhesion. Rat peritoneal macrophages were allowed to contact polymer substrates for periods ranging from 1 to 240 min before being subjected to a shear stress of 60-120 dynes/cm2 in a thin-channel flow cell. Percentage adhesion was calculated from the number of cells that remained adherent to the substrate after 30 s of applied shear stress. Macrophages remained adherent to 100% EMA and all hydroxystyrene-styrene copolymer surfaces after only 1 min of contact. In copolymers of the HEMA-EMA series, the time required to attain peak adhesion levels increased with increasing substrate hydrophilicity (increasing HEMA content). Cells did not attach to the 20% EMA/80% HEMA copolymer and the 100% HEMA polymer. The results demonstrate that there is a time delay between contact and adhesion of the cells to surfaces of increasing hydrophilicity within the HEMA-EMA series and no time delay with the hydroxystyrene-styrene series. The time delay is thought to be a function of the excluded volume provided by polymers that are able to undergo significant chain rotation and or swelling in the solvent, water. Small excluded volumes present in copolymers of high EMA content and all hydroxystyrene-styrene copolymers offer little or no resistance to formation of adhesive bonds by macrophages, whereas copolymers with large excluded volumes (high HEMA content) prevent contact and/or adhesion. A mechanism based on the net excluded volumes of both the cell and substrate surface macromolecule is proposed to explain this phenomenon.
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