From peptide precursors to oxazole and thiazole-containing peptide antibiotics: microcin B17 synthase.

Esherichia coli microcin B17 is a posttranslationally modified peptide that inhibits bacterial DNA gyrase. It contains four oxazole and four thiazole rings and is representative of a broad class of pharmaceutically important natural products with five-membered heterocycles derived from peptide precursors. An in vitro assay was developed to detect heterocycle formation, and an enzyme complex, microcin B17 synthase, was purified and found to contain three proteins, McbB, McbC, and McbD, that convert 14 residues into the eight mono- and bisheterocyclic moieties in vitro that confer antibiotic activity on mature microcin B17. These enzymatic reactions alter the peptide backbone connectivity. The propeptide region of premicrocin is the major recognition determinant for binding and downstream heterocycle formation by microcin B17 synthase. A general pathway for the enzymatic biosynthesis of these heterocycles is formulated.

Outbreak of Newcastle disease due to pigeon paramyxovirus type 1 in grey partridges (Perdix perdix) in Scotland in October 2006.

In October 2006, following an initially non-statutory disease investigation affecting 12-week-old grey partridges (Perdix perdix), an outbreak of Newcastle disease due to infection with the avian paramyxovirus type 1 virus responsible for the current panzootic in pigeons (PPMV-1) was confirmed in Scotland. Two pens of partridges were affected by signs including loss of condition, diarrhoea, progressive neurological signs and mortality totalling approximately 24 per cent, and laboratory evidence of the infection was obtained only in these groups. The premises had approximately 17,000 poultry including a collection of 375 birds of rare breeds, containing endangered breeds of significant conservation value, which were not culled but subjected to a health monitoring and testing programme. Investigations suggested that a population of feral pigeons living above the affected pens of partridges was the likely source of the outbreak. Laboratory and genetic analyses confirmed that the isolate recovered from the clinically affected partridges was PPMV-1, belonging to genetic lineage 4b. However, the virus could not be isolated from or detected in dead pigeons collected from the affected buildings.

In vivo processing and antibiotic activity of microcin B17 analogs with varying ring content and altered bisheterocyclic sites.

BACKGROUND: The Escherichia coli peptide antibiotic microcin B17 (MccB17) contains four oxazole and four thiazole rings, and inhibits DNA gyrase. The role of individual and tandem pairs of heterocycles in bioactivity has not been determined previously. RESULTS: The two tandem 4,2-bisheterocycles in MccB17 were varied by expression of MccB17 or mutants containing altered sequences at Gly39-Ser40-Cys41 or Gly54-Cys55-Ser56. A mixture of five-nine-ring MccB17 isoforms were separated and quantitated for antibiotic potency. Mutagenesis of the thiazole-oxazole pair significantly affected antibiotic activity compared with the upstream oxazole-thiazole, which might stabilize partially cyclized intermediates against proteolysis. CONCLUSIONS: Enzymatic heterocyclization in native MccB17 occurs distributively. Antibiotic activity correlates with the number of rings and is differentially sensitive to both the location and the identity of the 4,2-tandem heterocycle pairs in MccB17. Such tandem heterocycles might be useful pharmacophores in combinatorial libraries.

Open fractures of the pelvis. Review of 43 cases.

We reviewed 43 patients treated from 1984 to 1988 for open fractures of the pelvis. There were four Gustilo type I wounds, seven type II and 32 type III; 22 fractures were stable and 21 unstable. The overall mortality was 30%; the average Injury Severity Score was 30, being 26 in the survivors and 40 in the fetal cases. We analysed the influence of a number of factors on the mortality rate. The most important were the ISS and the age in years, while the presence of a type III wound and instability of the fracture also had an influence. We describe two simple methods of assessment of the prognosis in individual cases, based on these factors.

Articular surface partial-thickness rotator cuff tears.

Between 1987 and 1992, one hundred eleven articular surface partial-thickness rotator cuff tears were diagnosed in 106 patients; 90 were men, and 16 were women. The average age was 42.5 years. Average follow-up was 32.3 months, ranging from 26 to 84 months. Patients were separated into three groups. Group 1 consisted of 85 shoulders in which impingement was believed to be the primary cause; these shoulders were treated with debridement of the tear and arthroscopic subacromial decompression. Group 2 consisted of 14 shoulders with instability treated with debridement of the partial tear and anterior reconstruction (n = 10) or debridement and rehabilitation (n = 4). Group 3 contained 12 shoulders with tearing caused by trauma that were managed with debridement and open repair, if necessary. In 98 of 111 cases (88%), the patients had a satisfactory result. Five patients required open rotator cuff repair at a later date because of continued symptoms. Complications included significant postoperative stiffness in four shoulders, which required open release.

pH-dependent permeabilization of the plasma membrane of mammalian cells by anthrax protective antigen.

Protective antigen (PA) of anthrax toxin forms ion-conductive channels in planar lipid bilayers and liposomes under acidic pH conditions. We show here that PA has a similar permeabilizing action on the plasma membranes of CHO-K1 and three other mammalian cell lines (J774A.1, RAW264.7 and Vero). Changes in membrane permeability were evaluated by measuring the efflux of the K+ analogue, 86Rb+, from prelabelled cells, and the influx of 22Na+. The permeabilizing activity of PA was limited to a proteolytically activated form (PAN) and was dependent on acidic pH for membrane insertion (optimal at pH 5.0), but not for sustained ion flux. The flux was reduced in the presence of several known channel blockers: tetrabutyl-, tetrapentyl-, and tetrahexylammonium bromides. PAN facilitated the membrane translocation of anthrax edema factor under the same conditions that induced changes in membrane permeability to ions. These results indicate that PAN permeabilizes cellular membranes under conditions that are believed to prevail in the endosomal compartment of toxin-sensitive cells; and they provide a basis for more detailed studies of the relationship between channel formation and translocation of toxin effector moieties in vivo.

Cost of shoulder surgery.

A cost analysis of four common shoulder procedures was performed to better advise patients and payers of the expected costs and to provide a foundation for future cost/benefit analysis. Four groups of 50 patients each were evaluated. Group 1 underwent primary, open rotator cuff repair. Group 2 underwent open, anterior instability repair. Group 3 underwent arthroscopic subacromial decompression. Group 4 underwent hemiarthroplasty or total shoulder arthroplasty. The surgical and anesthetic fees, hospital bills, and perioperotive consultation charges were obtained and tabulated for all patients. The average ages for groups 1 to 4 were 56, 32, 50 and 63 years, respectively. In group 1 37% of the patients had worker's compensation, 35% had private insurance, and 28% had Medicare. In group 2 64% of the patients had worker's compensation, and 36% had private insurance. In group 3 39% of the patients had worker's compensation, 48% had private insurance, and 13% had Medicare. In group 4 4% of the patients had worker's compensation, 43% had private insurance, and 53% had Medicare. For group 1 the average length of stay was 3 days, and the average total cost was $9444. For group 2 the average stay was 1.7 days, and the average cost was $8675. For group 3 the average stay was 0 days, and the average cost was $7246. For group 4 the average stay was 5 days, and the average cost was $16,323. We believe that these figures represent the direct costs of four common types of shoulder surgery in a private setting and that such data are important to decision-making in a changing health care environment.

Effect of anthrax toxin's lethal factor on ion channels formed by the protective antigen.

Protective antigen (PA), a component of anthrax toxin, mediates translocation of the toxin's lethal and edema factors (LF and EF, respectively) to the cytoplasm, via a pathway involving their release from an acidic intracellular compartment. PA63, a 63-kDa proteolytic fragment of PA, can be induced to form ionconductive channels in the plasma membrane of mammalian cells by acidification of the medium. These channels are believed to be comprised of dodecyl sulfate-resistant oligomers (heptameric rings) of PA63 seen by electron microscopy of the purified protein. Here we report that the PA63-mediated efflux of 86Rb+ from preloaded CHO-K1 cells under acidic conditions is strongly inhibited (> or = 70%) by LF or LFN, a PA-binding fragment of LF. Control proteins caused no inhibition. Evidence is presented that the inhibition involves partial blockage of ion conductance by the PA63 channel. Also, oligomer formation is slowed somewhat by LF at pH values near the pH threshold of channel formation (pH approximately 5.3), suggesting that channel formation may also be retarded under these conditions. The relevance of these results to the location of the LF-binding site on PA63 and the mechanism of LF and EF translocation is discussed.

Closed wound drainage in shoulder surgery.

To evaluate the effectiveness of closed wound drainage in shoulder surgery, 300 patients were enrolled in a prospective randomized study. Three operations were studied: rotator cuff repair, anterior reconstruction for instability, and arthroplasty. One hundred patients were included in each group. All patients were evaluated for wound hematoma, infection, variation in postoperative rehabilitation caused by wound problems, and length of hospital stay. No statistical difference was found between the patients whose wounds were drained and those whose wounds were not drained. This finding existed within each category. Our data do not support the routine use of closed wound drainage in elective shoulder surgery.

Protective antigen-binding domain of anthrax lethal factor mediates translocation of a heterologous protein fused to its amino- or carboxy-terminus.

The edema factor (EF) and lethal factor (LF) components of anthrax toxin require anthrax protective antigen (PA) for binding and entry into mammalian cells. After internalization by receptor-mediated endocytosis, PA facilitates the translocation of EF and LF across the membrane of an acidic intracellular compartment. To characterize the translocation process, we generated chimeric proteins composed of the PA recognition domain of LF (LFN; residues 1-255) fused to either the amino-terminus or the carboxy-terminus of the catalytic chain of diphtheria toxin (DTA). The purified fusion proteins retained ADP-ribosyltransferase activity and reacted with antisera against LF and diphtheria toxin. Both fusion proteins strongly inhibited protein synthesis in CHO-K1 cells in the presence of PA, but not in its absence, and they showed similar levels of activity. This activity could be inhibited by adding LF or the LFN fragment (which blocked the interaction of the fusion proteins with PA), by adding inhibitors of endosome acidification known to block entry of EF and LF into cells, or by introducing mutations that attenuated the ADP-ribosylation activity of the DTA moiety. The results demonstrate that LFN fused to either the amino-terminus or the carboxy-terminus of a heterologous protein retains its ability to complement PA in mediating translocation of the protein to the cytoplasm. Besides its importance in understanding translocation, this finding provides the basis for constructing a translocation vector that mediates entry of a variety of heterologous proteins, which may require a free amino- or carboxy-terminus for biological activity, into the cytoplasm of mammalian cells.

Effect of ketorolac tromethamine (Toradol) on ecchymosis following anterior cruciate ligament reconstruction.

This article describes a study that assesses whether patients who received ketorolac tromethamine (Toradol; Syntex Research, Palo Alto, California) during knee surgery had an increased tendency to develop ecchymosis in the lower limb versus patients who did not receive ketorolac tromethamine. Sixty-four patients who underwent anterior cruciate ligament (ACL) surgery were divided randomly into three groups: patients who received Toradol at tourniquet inflation (TorTourn) at the end of surgery (TorEnd), or not at all (TorNone). None of the patients exhibited abnormal preoperative bleeding times. One week postsurgery, patients were evaluated photographically for ecchymosis between the hip and malleoli of the surgical limb. Ecchymotic areas between the hip and malleoli were traced around their borders with a black marker. Three photographs of each surgical knee were taken: posterior, anterolateral, and anteromedial views. Each patient's photos then were scanned into a computer and the amount of encircled (ecchymotic) surface area and the total surface area of the limb were calculated. For each view, the ecchymotic surface area was divided by the total surface area to obtain a percentage of ecchymosis on that view. The percentages for the three views were added to obtain a single score for each patient. The mean ecchymotic surface area score was 21.9 +/- 31% for the TorTourn group, 27.5 +/- 25.5% for the TorEnd group, and 30.3 +/- 36.4% for the TorNone group. There was no significant difference in the ecchymotic surface area among the groups. This study suggests that ketorolac tromethamine does not affect the amount of ecchymosis that occurs following knee surgery.

Anthrax protective antigen forms oligomers during intoxication of mammalian cells.

The protective antigen component (PA) of anthrax toxin binds to receptors on target cells and conveys the toxin's edema factor (EF) and lethal factor (LF) components into the cytoplasm. PA (83 kDa) is processed by a cellular protease, yielding a 63-kDa fragment (PA63), which binds EF and/or LF. When exposed to acidic pH, PA63 inserts into membranes and forms ion-conductive channels. By electron microscopy, a significant fraction of purified PA63 was found to be in the form of a multi-subunit ring-shaped oligomer (outer diameter, 10.4 nm). The rings are heptameric, as judged by inspection and by rotational power spectra. Purified PA63 showed a high M(r) band, apparently corresponding to the oligomer, on SDS-polyacrylamide gels, and oligomer of similar size was formed in cells in a time-dependent manner after addition of full-length PA. Inhibitors of internalization and endosome acidification blocked conversion of cell-associated PA to a high molecular weight species, and medium at pH 5.0 induced oligomer formation in the presence or absence of the inhibitors. These results correlate PA63 oligomerization with conditions required for translocation of EF and LF across lipid bilayers, implying that the PA63 oligomer may function in translocation.

ATP/GTP hydrolysis is required for oxazole and thiazole biosynthesis in the peptide antibiotic microcin B17.

In the maturation of the Escherichia coli antibiotic Microcin B17, the product of the mcbA gene is modified posttranslationally by the multimeric Microcin synthetase complex (composed of McbB, C, and D) to cyclize four Cys and four Ser residues to four thiazoles and four oxazoles, respectively. The purified synthetase shows an absolute requirement for ATP or GTP in peptide substrate heterocyclization, with GTP one-third as effective as ATP in initial rate studies. The ATPase/GTPase activity of the synthetase complex is conditional in that ADP or GDP formation requires the presence of substrate; noncyclizable versions of McbA bind to synthetase, but do not induce the NTPase activity. The stoichiometry of ATP hydrolysis and heterocycle formation is 5:1 for a substrate that contains two potential sites of modification. However, at high substrate concentrations (>50Km) heterocycle formation is inhibited, while ATPase activity occurs undiminished, consistent with uncoupling of NTP hydrolysis and heterocycle formation at high substrate concentrations. Sequence homology reveals that the McbD subunit has motifs reminiscent of the Walker B box in ATP utilizing enzymes and of motifs found in small G protein GTPases. Mutagenesis of three aspartates to alanine in these motifs (D132, D147, and D199) reduced Microcin B17 production in vivo and heterocycle formation in vitro, suggesting that the 45 kDa McbD has a regulated ATPase/GTPase domain in its N-terminal region necessary for peptide heterocyclization.

Fused polycationic peptide mediates delivery of diphtheria toxin A chain to the cytosol in the presence of anthrax protective antigen.

The lethal factor (LF) and edema factor (EF) of anthrax toxin bind by means of their amino-terminal domains to protective antigen (PA) on the surface of toxin-sensitive cells and are translocated to the cytosol, where they act on intracellular targets. Genetically fusing the amino-terminal domain of LF (LFN; residues 1-255) to certain heterologous proteins has been shown to potentiate these proteins for PA-dependent delivery to the cytosol. We report here that short tracts of lysine, arginine, or histidine residues can also potentiate a protein for such PA-dependent delivery. Fusion of these polycationic tracts to the amino terminus of the enzymic A chain of diphtheria toxin (DTA; residues 1-193) enabled it to be translocated to the cytosol by PA and inhibit protein synthesis. The efficiency of translocation was dependent on tract length: (LFN > Lys8 > Lys6 > Lys3). Lys6 was approximately 100-fold more active than Arg6 or His6, whereas Glu6 and (SerSerGly)2 were inactive. Arg6DTA was partially degraded in cell culture, which may explain its low activity relative to that of Lys6DTA. The polycationic tracts may bind to anionic sites at the cell surface (possibly on PA), allowing the fusion proteins to be coendocytosed with PA and delivered to the endosome, where translocation to the cytosol occurs. Excess free LFN blocked the action of LFNDTA, but not of Lys6DTA. This implies that binding to the LF/EF site is not an obligatory step in translocation and suggests that the polycationic tag binds to a different site. Besides elucidating the process of translocation in anthrax toxin, these findings may aid in developing systems to deliver heterologous proteins and peptides to the cytoplasm of mammalian cells.

Cofactor requirements and reconstitution of microcin B17 synthetase: a multienzyme complex that catalyzes the formation of oxazoles and thiazoles in the antibiotic microcin B17.

In the maturation of the Escherichia coli antibiotic Microcin B17 (MccB17), the McbA prepro-antibiotic is modified post-translationally by the multimeric microcin synthetase complex (composed of the McbB, -C, and -D proteins), which cyclizes four cysteines and four serines to thiazoles and oxazoles, respectively. Herein, we report the purification of individual subunits of MccB17 synthetase as fusions to maltose binding protein (MBP), and the in vitro reconstitution of heterocyclization activity. Preliminary characterization of each subunit reveals McbB to be a zinc-containing protein that may catalyze the initial cyclodehydration step, and McbC to contain flavin, consistent with an anticipated role for a dehydrogenase. We have previously demonstrated that McbD is a regulated ATPase/GTPase that may function as a conformational switch. Photolabeling experiments with the McbA propeptide now identify McbD as the initial site of substrate recognition. Heterocyclization activity was reconstituted only by combining all three subunits, demonstrating that each protein is required for heterocycle formation. Titration assays indicate that the subunits bind to each other with at least micromolar affinities, although McbD affords activity only after the MBP tag is proteolytically removed. Subunit competition assays with an McbDD147A mutant, which yields a catalytically deficient synthetase in vivo, show it to be defective in complex formation, whereas the McbBC181A/C184A double mutant, which is also inactive, competitively inhibits reconstitution by native McbB. Addition of the HtpG chaperone (originally shown to copurify with MccB17 synthetase), does not stimulate synthetase reconstitution or heterocyclization activity in vitro. A model for synthetase activity is proposed.