ABSTRACT
The cathelicidin antimicrobial peptide bactenecin is a beta-hairpin molecule with a single disulfide bond and broad antimicrobial activity. The proform of bactenecin exists as a dimer, however, and it has been proposed that bactenecin is released as a dimer in vivo, although there has been little study of the dimeric form of bactenecin. To investigate the effect of bactenecin dimerization on its biological activity, we characterized the dimer's effect on phospholipid membranes, the kinetics of its bactericidal activity, and its salt sensitivity. We initially synthesized two bactenecin dimers (antiparallel and parallel) and two monomers (beta-hairpin and linear). Under oxidative folding conditions, reduced linear bactenecin preferentially folded into a dimer forming a ladder-like structure via intermolecular disulfide bonding. As compared to the monomer, the dimer had a greater ability to induce lysis of lipid bilayers and was more rapidly bactericidal. Interestingly, the dimer retained antimicrobial activity at physiological salt concentrations (150 mm NaCl), although the monomer was inactivated. This salt resistance was also seen with bactenecin dimer containing one intermolecular disulfide bond, and the bactenecin dimer appears to undergo multimeric oligomerization at high salt concentrations. Overall, dimeric bactenecin shows potent and rapid antimicrobial activity, and resists salt-induced inactivation under physiological conditions through condensation and oligomerization. These characteristics shed light on the features that a peptide would need to serve as an effective therapeutic agent.
Subject(s)
Anti-Bacterial Agents/chemistry , Peptides, Cyclic/chemistry , Sodium Chloride/chemistry , Amino Acid Sequence , Anti-Bacterial Agents/isolation & purification , Anti-Bacterial Agents/pharmacology , Circular Dichroism , Dimerization , Hemolysis/drug effects , Kinetics , Liposomes , Molecular Sequence Data , Peptides, Cyclic/isolation & purification , Peptides, Cyclic/pharmacology , Protein Conformation , Protein FoldingABSTRACT
Botulinum toxin type A was intramuscularly administered to Sprague-Dawley rats once a day for 28 days at doses of 1, 3, and 9 ng kg-1 day-1 to investigate the possibility of unanticipated toxicity of repeated dose. A dose-related decrease in body weight gain was noted and lasted throughout the 4-week recovery period. Paralytic gait was a common clinical sign observed in the animals dosed at >or=3 ng kg-1 day-1 and muscle atrophy at 9 ng kg-1 day-1. Decreased creatinine was monitored in both males and females treated at 9 ng kg-1 day-1. Microscopic examination of the quadriceps femoris muscle, the test article application site, confirmed the muscle atrophy with a decrease in myofiber diameter and an increase of myofiber nuclei and intermyofiber connective tissue. Although antibody against botulinum toxin type A was detected in the sera from both males and females at 9 ng kg-1 day-1, no immunogenicity-related changes or lesions were noted. In conclusion, no other side effects of the botulinum toxin type A injection except the decrease in body weight gain and the muscle atrophy at the administration site were noted in the 28-day intramuscular repeated dose study.
