Biomedical subjects

J D Radolf

Publications and source records attributed to J D Radolf.

113 records · Page 7Linked to original sources

PubMed1986

Native surface association of a recombinant 38-kilodalton Treponema pallidum antigen isolated from the Escherichia coli outer membrane.

A recombinant plasmid designated pAW305, containing a 6-kilobase insert of Treponema pallidum DNA, directed the expression of a 38-kilodalton (kDa) treponemal antigen in Escherichia coli. The 38-kDa antigen copurified with the outer membrane fraction of the E. coli cell envelope after treatment with nonionic detergents or sucrose density gradient centrifugation. Rabbits immunized with the recombinant 38-kDa antigen developed antibodies which reacted specifically with a 38-kDa T. pallidum antigen on immunoblots, and 38-kDa antisera specifically immobilized T. pallidum in a complement-dependent manner in the T. pallidum immobilization test. Antisera to the 38-kDa recombinant antigen were also used to demonstrate its native surface association on T. pallidum by immunoelectron microscopy.

Animals↗

PubMed1986

Antigenic interrelationship between endoflagella of Treponema phagedenis biotype Reiter and Treponema pallidum (Nichols): molecular characterization of endoflagellar proteins.

Purified endoflagella from nonpathogenic Treponema phagedenis biotype Reiter were characterized biochemically and compared antigenically with the endoflagellar proteins of Treponema pallidum. T. phagedenis biotype Reiter endoflagella were dissociated into constituent polypeptides by incubation under conditions which disrupt noncovalent bonds. Chymotrypsin peptide maps of T. phagedenis biotype Reiter endoflagellar proteins revealed that the 37- and 33-kilodalton (kDa) major components shared significant homology with the 27- and 30-kDa minor components, respectively. The peptide maps also suggested that the major components shared a lesser degree of structural similarity with each other. These relationships were confirmed by Western blots of T. phagedenis biotype Reiter endoflagellar proteins employing antibodies that were purified against the individual endoflagellar polypeptides. Western blots of T. pallidum with the purified antibodies also demonstrated strong cross-reactivity between the T. phagedenis biotype Reiter endoflagellar proteins and T. pallidum proteins of identical or similar molecular weights. A unique Western blotting technique that we called epitope bridging was used to determine that the 37-kDa subunit contains most of the external epitopes on T. phagedenis biotype Reiter endoflagella. Immunoelectron microscopy with human syphilitic serum and rabbit T. phagedenis biotype Reiter endoflagellar antiserum confirmed the presence of cross-reactive epitopes on the surface of intact T. phagedenis and T. pallidum endoflagella.

Animals↗

PubMed1986

Properties of an ordered ring structure formed by recombinant Treponema pallidum surface antigen 4D.

Ultrastructural and biochemical studies of a recombinant Treponema pallidum surface antigen designated 4D have been conducted due to its likely biological significance. Electron microscopy demonstrated that the 190-kilodalton (kDa) 4D molecule is an ordered ring structure of 10-nm diameter. The 90-kDa proteinase K-treated 4D is an ordered ring structure of 6-nm diameter. Evidence is presented that the 190-kDa ordered ring is maintained by noncovalent bonds; 19-kDa monomers can reassociate in vitro to reform a 190-kDa molecule. Amino acid composition analysis of 190-kDa 4D showed that the molecule is composed of 45% hydrophobic residues. Evidence relating the structure of the 4D ordered ring to its potential role in the pathogenesis of syphilis is discussed.

Amino Acids↗

PubMed1984

Purification and characterization of a cloned protease-resistant Treponema pallidum-specific antigen.

A cloned Treponema pallidum antigen, designated 4D, was purified from Escherichia coli predominantly as a 190-kilodalton (kd) polypeptide, although higher oligomeric forms exist. Extensive proteolysis of 4D created a limit digestion product of 90 kd which retained antigenicity with sera from patients with primary, secondary, early latent, late latent, and tertiary syphilis. A molecule indistinguishable from 90-kd 4D in size, isoelectric point, and antigenicity was isolated from T. pallidum after proteolysis. The 190- and 90-kd forms of 4D were stable at 68 degrees C but converted to 19- and 14-kd species, respectively, after boiling in sodium dodecyl sulfate. The low-molecular-weight species did not react with syphilitic sera. Rabbits immunized with the purified 4D antigen developed antibodies which immobilized virulent T. pallidum in a complement-dependent assay system, suggesting that the antigen has a native surface location.

Animals↗

PubMedDate not supplied

Solid-phase synthesis of biologically active lipopeptides as analogs for spirochetal lipoproteins.

Bacterial lipoproteins, which are of particular interest because of their immunomodulatory activities, share a common N-terminal structural motif that consists of an N-acyl-S-diacylglyceryl cysteine residue. Synthetic tripalmitoylated analogs of the N-terminal sequences of several bacterial lipopetides have been found to reproduce the immunological activities of the corresponding intact lipoproteins. Methods for the synthesis of lipopeptide analogs of bacterial lipoproteins have hitherto relied upon the coupling of peptide moieties, lacking the N-terminal cystienyl residue, with a tripalmitoylglyceryl cysteine moiety synthesized separately in solution. A method is described here by which rapid and convenient synthesis of the entire lipopeptide is accomplished by solid-phase methods in which the N-terminal cysteinyl derivative is assembled stepwise while attached to the completed peptide moiety prior to cleavage from the resin. The method has been used to synthesize two lipohexapeptides representing the N-terminal sequences of the 47-kDa membrane lipoprotein of the syphilis spirochete, Treponema pallidum, and the outer surface protein A (OspA) of the Lyme disease spirochete, Borrelia burgdorferi. These lipopeptides, which were synthesized without detectable endotoxin contamination, exhibit macrophage-stimulating activity that is not expressed by the corresponding non-acylated hexapeptides. The data indicate that synthetic lipopeptides based on spirochetal lipoproteins are appropriate substitutes for the intact lipoproteins in immunological studies.

Amino Acid Sequence↗