Microcin 7 is a small peptide produced and excreted to the culture medium by stationary-phase Escherichia coli cells harboring the pMccC7 plasmid (formerly named pRYC7). This peptide inhibited the growth of the enterobacteria phylogenetically closer to E. coli, apparently by blocking protein biosynthesis. The molecule was degraded with trypsin, and the resulting fragments were purified and sequenced. The results show that microcin 7 is a linear heptapeptide blocked at both ends.Microcins are dialyzable (i.e., low-molecular-weight) antibacterial agents originally found in bacterial isolates from the feces of newborn infants (1). Most of the microcins seem to be peptides, and, unlike the classical bacteriocins, they are not inducible by agents that trigger the SOS system for DNA repair (for a review, see reference 4).The synthesis of microcins is not lethal for the producer cells, and it requires the presence of plasmids specific for each type of microcin (3,21). Five such microcin types have been characterized so far. In each case, the microcinogenic plasmid is also implicated in the expression of the immunity that the producer strains show to their own microcins, even when the microcins are added exogenously (4).Free microcin-like substances have been found in fecal samples, and microcin-producing strains tend to predominate in patients with jejunal bacterial overgrowth. Furthermore, the colonization ability of Escherichia coli K-12 given orally to Swiss mice is clearly enhanced by microcinogenic plasmids (4). These results, together with the high diffusibility and in some cases the resistance of microcins to intestinal proteases, have led us to propose a role for these compounds in the displacements of bacterial populations that take place in the intestine under both normal and pathological conditions (2). Bacteriocins are no longer thought to play a role in such ecological successions (15), but as we mentioned above, microcins do not look like classical bacteriocins. As a matter of fact, they share properties with both bacteriocins and the secondary-metabolism antibiotics.These peculiar features of microcins and their presence in the human intestinal ecosystem drew our attention to these substances, and we chose microcin 7 (or C7 according to the last proposals [4]) for further studies. Since there were only scarce and indirect data on the chemical structure of microcins, we undertook the purification and analysis of microcin 7. The molecule was purified from the supematants of stationary cultures of E. coli RYC25 and was found to contain seven different amino acids at the most, which accounted for the apparent molecular weight as estimated by gel filtration chromatography (11).In the present work we report the amino acid sequence of the peptide (hence the first structural information on a microcin), the spectrum of its antimicrobial activity, and the first data on its mode of action. MATERIALS AND METHODSTrypsin treated with diphenylcarbamyl chloride, carboxypeptidase Y, phenylthiohydantoin amino acids, and P...
Pro‐urokinase is a natural plasminogen activator that displays a clot‐lysis activity through a fibrin‐dependent mechanism. It seems to be a promising agent for the treatment of coronary thrombosis. Like tissue‐type plasminogen activator and two‐chain urokinase‐type plasminogen activator, pro‐urokinase has a very short half‐life in circulation. It has been described that conjugation of serum albumin with pro‐urokinase in plasma may occur that could protect this protein from degradation. In this study we describe the insertion of an extra cysteine residue in the N‐terminal end of des‐(C11–K135)‐pro‐urokinase (Δ125‐proUK), a pro‐urokinase deletion mutant lacking amino acids 11–135. We have expressed and purified the new mutein [H5K, S9C, N10T]des‐(C11‐K135)‐pro‐urokinase (Cys‐Δ125‐pro‐urokinase) and chemically conjugated it with serum albumin via the extra cysteine of Cys‐Δ125‐pro‐urokinase. The purified conjugate obtained has a lower specific amidolytic activity (72000 U/mg) than unconjugated Cys‐Δ125‐pro‐urikinase (240000 U/mg) due to its higher molecular mass and has a similar fibrinolytic activity in a clot lysis test to that of Δ125‐pro‐urokinase. We established an ELISA to measure the concentration of the conjugate in plasma and to follow the pharmacokinetics of the conjugate in monkeys after bolus injection. The conjugate displays significant lysis of human plasma clots in vivo and a dramatic increase of the half‐life in the circulation, with respect to pro‐urokinase and Δ125‐pro‐urokinase. Therefore, preliminary biological characterisation of this conjugate indicates that it could be a good candidate to inject as a bolus, compared with the infusion regimen needed with pro‐urokinase.
Pro-urokinase is a natural plasminogen activator that displays a clot-lysis activity through a fibrin-dependent mechanism. It seems to be a promising agent for the treatment of coronary thrombosis. Like tissue-type plasminogen activator and two-chain urokinase-type plasminogen activator, pro-urokinase has a very short half-life in circulation. It has been described that conjugation of serum albumin with pro-urokinase in plasma may occur that could protect this protein from degradation. In this study we describe the insertion of an extra cysteine residue in the N-terminal end of des-(C11-K135)-pro-urokinase (delta 125-proUK), a pro-urokinase deletion mutant lacking amino acids 11-135. We have expressed and purified the new mutein [H5K, S9C, N10T] des-(C11-K135)-pro-urokinase (Cys-delta 125-pro-urokinase) and chemically conjugated it with serum albumin via the extra cysteine of Cys-delta-pro-urokinase. The purified conjugate obtained has a lower specific amidolytic activity (72,000 U/mg) than unconjugated Cys-delta 125-pro-urikinase (240,000 U/mg) due to its higher molecular mass and has a similar fibrinolytic activity in a clot lysis test to that of delta 125-pro-urokinase. We established an ELISA to measure the concentration of the conjugate in plasma and to follow the pharmacokinetics of the conjugate in monkeys after bolus injection. The conjugate displays significant lysis of human plasma clots in vivo and a dramatic increase of the half-life in the circulation, with respect to pro-urokinase and delta 125-pro-urokinase. Therefore, preliminary biological characterisation of this conjugate indicates that it could be a good candidate to inject as a bolus, compared with the infusion regimen needed with pro-urokinase.
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