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.     

Analysis of fluoromethyl group chirality establishes a common stereochemical course for the enolpyruvyl transfers catalyzed by EPSP synthase and UDP-GlcNAc enolpyruvyl transferase

The stereochemistry of transient methyl group formation at C-3 of phosphoenolpyruvate (PEP) in the reaction catalyzed by 5-enolpyruvylshikimate 3-phosphate (EPSP) synthase has been examined using the pseudosubstrates, (E)- and (Z)-3-fluorophosphoenolpyruvate (FPEP). Kinetically stable, chiral [1H, 2H]fluoromethyl analogs of the reaction tetrahedral intermediate were isolated and subjected to decomposition and stereochemical analysis. EPSP synthase was found to catalyze the 2-re face addition of solvent-derived hydrogen to C-3 of FPEP (corresponding to the 2-si face of PEP). Comparison of these data with prior analogous work on the MurA reaction [Kim, D.H., Lees, W.J., & Walsh, C. T. (1995) J. Am. Chem. Soc. 117, 6380-6381] suggests that the two enolpyruvyl transferases share a common stereochemical course, further strengthening the mechanistic, structural, and evolutionary relationship between the two enzymes.     

Dynamic axial external fixation in the surgical treatment of tibia vara

Osteotomy is the well-established treatment of Blount's disease (tibia vara), although the types of fixation used vary considerably. The use of dynamic axial external fixation to stabilize osteotomies for tibia vara until solid union occurs without the use of supplemental casting has not been reported by other authors. From 1985 until the present, we have used osteotomy with dynamic axial external fixation as treatment of 31 tibiae in 23 patients. All osteotomies healed and there was no postoperative loss of correction. There was an average correction of 20 degrees between the pre- and postoperative mechanical axis. Advantages of dynamic axial external fixation include ease of application, adjustability, early weight bearing, the ability to lengthen the extremity, and no second operation for removal of hardware. Based on our results, we believe that dynamic axial external fixation is an excellent form of osteotomy stabilization in the surgical treatment of tibia vara.     

Neonatology radiology casebook. Tracheal agenesis

BACKGROUND: Biliary tract diseases are frequent in heart transplant recipients, with significant morbidity and mortality. Since the first presentation of gallstones in this population is often acute cholecystitis, asymptomatic cholelithiasis should not be considered benign. PATIENTS AND METHODS: We retrospectively reviewed 18 heart transplant recipients who underwent cholecystectomy from January 1991 to June 1997. We intentionally chose to perform a straightforward open procedure when acute cholecystitis was suspected (3 patients). A laparoscopic cholecystectomy was performed in all the other cases (15 patients) without conversion to open procedure. CONCLUSION: Since no significant complications were observed in our patients, we believe that transplant recipients with cholelithiasis should undergo laparoscopic cholecystectomy in their posttransplantation course regardless of the symptomatic status of their biliary tract.     

Knowledge and attitudes of health-care providers toward cancer pain management: a comparison of physicians, nurses, and pharmacists in the state of New Hampshire

The knowledge and attitudes toward cancer pain management of physicians, nurses, and pharmacists in the state of New Hampshire were examined through the use of a statewide survey. Many of the providers who completed the survey, and thus indicated that they treated patients with cancer pain on a regular basis, were not pain or oncology specialists. Most of these providers were quite well informed about the fundamentals of cancer pain management. Approximately 90% of providers in all three groups were not concerned about addiction among cancer patients. Yet, there was a small percentage of providers who responded in less than optimal ways to items dealing with opioid pharmacology, pain assessment, and the importance of pain relief. Comparison of responses among provider groups indicated that nurses were the most knowledgeable and pharmacists the least knowledgeable about pain assessment. Physicians were the most knowledgeable regarding opioid pharmacology but seemed the least committed to providing optimal pain relief. Further analysis identified a small group of physicians that included a disproportionately high percentage of family practitioners and surgeons who consistently responded in less than optimal ways to items dealing with the importance of pain relief. The results of this study indicate a continuing need for broad-based educational programs in cancer pain management and for new initiatives focused on practitioners who see relatively few cancer patients and may have difficulty accessing traditional educational programs.     

Effect of ambulatory utilization review on referrals from generalists to specialists

We studied whether ambulatory utilization review (UR) alters how many patients internal medicine residents refer to sub-specialists, and whether the effect persists without reinforcement. We compared referral rates of residents from a firm that held UR meetings (intervention firm residents, n = 20) with those of residents from a firm that did not (control firm residents, n = 21). We then compared referral rates of 17 intervention firm residents while they were participating in UR with their rates after not participating for at least 4 weeks. Intervention firm residents submitted 30% fewer referrals than control firm residents (9% vs 13%, p = .05). However, the effect was short-lived; after 4 weeks without UR, intervention firm resident referral rates were similar to control firm referral rates.     

Correcting the bias of clerkship timing on academic performance

OBJECTIVES: To document the time-of-year bias in National Board of Medical Examiners subject examination (NBME) scores in a third-year pediatrics clerkship and to develop a grading method that neutralizes the bias. DESIGN: Interventional modeling of final grades. SETTING: University-based medical school. SUBJECTS AND METHODS: During each of the past 3 academic years, we conducted six 2-month pediatric clerkships for third-year students. To counter the time-of-year bias, NBME scores, clinical evaluations, and departmental examination scores for the current rotation were pooled with those from the rotations from the same time of year during the previous 2 years. Final grades for the current rotation were determined by cutoff points derived from that entire 3-year pool. We analyzed this approach by testing the time-of-year effects on NBME scores, clinical evaluations, and final grades while maintaining step 1 of the US Medical Licensing Examination as a preclinical baseline control. RESULTS: The scores for step 1 of the US Medical Licensing Examination did not differ significantly by time of year. Clinical evaluations and NBME scores showed significant upward trends as the academic year progressed. By contrast, according to design, final grades showed no significant time-of-year trend. CONCLUSIONS: Our results support previous reports of significant improvements in NBME scores across the academic year. Our method of computing final grades corrects for this time-of-year bias by judging students only in relation to those who took the rotation at the same time of year. It is our belief that the prevalence and significance of the time-of-year trend warrants such an adjustment in grading to help minimize the academic disadvantage faced by students early in their clinical training.     

Selective convective brain cooling during normothermic cardiopulmonary bypass in dogs

BACKGROUND: Neurologic complications, primarily resulting from ischemic insults, represent the leading cause of morbidity and disability, and the second most common source of death, after cardiac operations. Previous studies have reported that increases (as occur during the rewarming phase of cardiopulmonary bypass [CPB]) or decreases in brain temperature of a mere 0.5 degrees to 2 degrees C can significantly worsen or improve, respectively, postischemic neurologic outcome. The purpose of the present study was to evaluate a novel approach of selectively cooling the brain during hypothermic CPB and subsequent rewarming. METHODS: Sixteen dogs were anesthetized with either intravenous pentobarbital or inhaled halothane (n = 8 per group). Normocapnia (alpha stat technique) and a blood pressure near 75 mm Hg were maintained. Temperatures were monitored by placing thermistors in the esophagus (i.e., core), parietal epidural space, and brain parenchyma at depths of 1 and 2 cm beneath the dura. During CPB, core temperature was actively cycled from 38 degrees C to 28 degrees C, and then returned to 38 degrees C. Forced air pericranial cooling (air temperature of approximately 13 degrees C) was initiated simultaneous with the onset of CPB, and maintained throughout the bypass period. Brain-to-core temperature gradients were calculated by subtracting the core temperature from regional brain temperatures. RESULTS: In halothane-anesthetized dogs, brain temperatures at all monitoring sites were significantly less than core during all phases of CPB, with one exception (2 cm during systemic cooling). Brain cooling was most prominent during and after systemic rewarming. For example, during systemic rewarming, average temperatures in the parietal epidural space, and 1 and 2 cm beneath the dura, were 3.3 degrees +/- 1.3 degrees C (mean +/- standard deviation), 3.2+/-1.4 degrees C, and 1.6 degrees +/-1.0 degrees C, cooler than the core, respectively. Similar trends, but of a greater magnitude, were noted in pentobarbital-anesthetized dogs. For example, during systemic rewarming, corresponding brain temperatures were 6.5 degrees +/-1.7 degrees C, 6.3 degrees +/-1.6 degrees C, and 4.2+/-1.3 degrees C cooler than the core, respectively. CONCLUSIONS: The magnitude of selective brain cooling observed in both study groups typically exceeded the 0.5 degrees to 2.0 degrees C change previously reported to modulate ischemic injury, and was most prominent during the latter phases of CPB. When compared with previous research from our laboratory, application of cold forced air to the cranial surface resulted in brain temperatures that were cooler than those observed during hypothermic CPB without pericranial cooling. On the basis of the assumption that similar beneficial brain temperature changes can be induced in humans, we speculate that selective convective brain cooling may enable clinicians to improve neurologic outcome after hypothermic CPB.