ABSTRACT
Two 99Tcm-labelled analogues of the chemotactic peptide ForMLF were evaluated as potential agents for imaging inflammation and infection, in the hope that they would be simple to use and would give diagnostically useful images shortly after injection. The peptides differed in the chelation site for 99Tcm and the presence of a hydrophilic spacer. The sequences of RP050 and RP056 were ForNleLFNleYK(G)G-C(Acm)-GPic and ForNleLFNleYKK(DG)GC(Acm)SPic respectively, where Pic is picolinic acid. In in vitro tests of binding to the ForMLF receptor on polymorphonuclear neutrophils and potency for release of myeloperoxidase, RP056 was similar in potency to ForMLF, whereas RP050 was 10 times more potent. When administered in 5-nmol doses to rats, RP050 produced less extensive neutropenia than ForMLF, whereas RP056 produced very little neutropenia. Following labelling by ligand exchange from tartrate or glucoheptonate at 100 degrees C and purification using a C-18 solid-phase extraction cartridge, 4-MBq doses were administered to rats bearing infectious (Escherichia coli) or sterile (zymosan) inflammation sites in the thigh. The inflammation-to-normal muscle ratios at 30 min after injection were 3.9 +/- 0.4 for RP050 and 4.7 +/- 0.3 for RP056 (mean +/- S.E.M., n = 4), and the ratios were maintained for up to 3 h. These peptides are promising agents for imaging inflammation and infection.
Subject(s)
Chemotactic Factors , Inflammation/diagnostic imaging , Oligopeptides , Technetium , Amino Acid Sequence , Animals , Chelating Agents/adverse effects , Chelating Agents/chemistry , Chemotactic Factors/adverse effects , Chemotactic Factors/chemistry , In Vitro Techniques , Male , Molecular Sequence Data , N-Formylmethionine Leucyl-Phenylalanine/analogs & derivatives , N-Formylmethionine Leucyl-Phenylalanine/chemistry , Neutropenia/etiology , Neutropenia/prevention & control , Oligopeptides/adverse effects , Oligopeptides/chemistry , Radionuclide Imaging , Rats , Rats, Sprague-Dawley , Receptors, Formyl Peptide , Receptors, Immunologic/metabolism , Receptors, Peptide/metabolism , Technetium/adverse effectsABSTRACT
Bacillus subtilis NRRL 365 produced high extracellular carboxyl esterase activity in submerged culture media containing wheat bran, corn steep liquor, and salts. Supplementation of this medium with glucose reduced esterase activity to 37% of that in the unsupplemented control. Esterase activity was purified by ammonium sulfate fractionation, DEAE-Sephadex A-50 ion-exchange chromatography with sodium chloride gradient elution, and preparative polyacrylamide gel electrophoresis. The resultant purified components, esterases I and II, manifested single bands following silver staining of polyacrylamide gel electrophoresis gels and had final specific activities of 80 and 520 U/mg, respectively. Molecular weights for components I and II were 36,000 and 105,000 to 110,000, respectively. Esterases I and II both had a pH optimum of 8.0, with relative activities of 10 and 85%, respectively, at pH 9.0. K(m)s with p-nitrophenylacetate were 0.91 mM for esterase I and 0.67 mM for esterase II. In general, patterns of enzyme inhibition were similar for both components. Differences were observed in the relative activities of esterases I and II towards p-nitrophenyl esters of acetate, propionate, and butyrate; Activity ratios for components I and II were 100:94:48 and 100:36:23, respectively. The purified components did not hydrolyze long-chain triglycerides and did not manifest proteolytic activity.
