Stereochemical course of enzymatic enolpyruvyl transfer and catalytic conformation of the active site revealed by the crystal structure of the fluorinated analogue of the reaction tetrahedral intermediate bound to the active site of the C115A mutant of MurA

MurA (UDP-GlcNAc enolpyruvyl transferase), the first enzyme in bacterial peptidoglycan biosynthesis, catalyzes the enolpyruvyl transfer from phosphoenolpyruvate (PEP) to the 3'-OH of UDP-GlcNAc by an addition-elimination mechanism that proceeds through a tetrahedral ketal intermediate. The crystal structure of the Cys115-to-Ala (C115A) mutant of Escherichia coli MurA complexed with a fluoro analogue of the tetrahedral intermediate revealed the absolute configuration of the adduct and the stereochemical course of the reaction. The fluorinated adduct was generated in a preincubation of wild-type MurA with (Z)-3-fluorophosphoenolpyruvate (FPEP) and UDP-GlcNAc and purified after enzyme denaturation. The fluorine substituent stabilizes the tetrahedral intermediate toward decomposition by a factor of 10(4)-10(6), facilitating manipulation of the adduct. The C115A mutant of MurA was utilized to avoid the microheterogeneity that arises in the wild-type MurA from the attack of Cys115 on C-2 of FPEP in competition with the formation of the fluorinated adduct. The crystal structure of the complex was determined to 2.8 A resolution, and the absolute configuration at C-2 of the adduct was found to be 2R. Thus, addition of the 3'-OH of UDP-GlcNAc is to the 2-si face of FPEP, corresponding to the 2-re face of PEP. Given the previous observation that, in D2O, the addition of D+ to C-3 of PEP proceeds from the 2-si face [Kim, D. H., Lees, W. J., and Walsh, C. T. (1995) J. Am. Chem. Soc. 117, 6380-6381], the addition across the double bond of PEP is anti. Also, because the overall stereochemical course has been shown to be either anti/syn or syn/anti [Lees, W. J., and Walsh, C. T. (1995) J. Am. Chem. Soc. 117, 7329-7337], it now follows that the stereochemistry of elimination of H+ from C-3 and Pi from C-2 of the tetrahedral intermediate of the reaction is syn.     

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.     

Structure of the 3'-hairpin of the TYMV pseudoknot: preformation in RNA folding

The solution structure of an RNA-hairpin present in the pseudoknot, which is found at the 3'-terminus of turnip yellow mosaic virus genomic RNA, has been solved by nuclear magnetic resonance spectroscopy. The loop, which contains the sequence 5'-GGGUCA-3', was found to be highly structured and, contrary to expectations, does not attain its stability through GA or GC base pair formation but by triple interactions between the tilted adenosine and the minor groove sides of the first two guanosines. Interestingly, a very similar conformation was found for the cognate pseudoknot, implying that the 3'-hairpin is preformed for folding into a pseudoknotted structure. These findings suggest a mechanism of 'predetermined-fit' as a principle in RNA folding.     

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.     

The threat of funding cuts for graduate medical education: survey of decision makers

OBJECTIVE: To assess the potential actions of medical school deans, graduate medical education (GME) committee chairs, and hospital chief executive officers (CEOs) regarding future funding reductions for residency training. Specifically, institutions with emergency medicine (EM) residencies were surveyed to see whether EM training was disproportionally at risk for reductions. METHODS: An anonymous 2-page survey was used. Ninety-eight EM residency programs were identified using the American Medical Association Graduate Medical Education Directory 1994-95. Seventy deans, 102 GME chairs, and 97 hospital CEOs were identified. The survey posed a hypothetical 25% forced reduction in residency positions and asked the decision makers for their responses. Options included: 1) proportional reductions of training positions from all residencies, 2) proportional reductions in either primary care or specialty residency positions, or 3) reduction or elimination of specific training programs. The survey asked for a first and second choice of residencies to be reduced or eliminated from an alphabetical list of 17. The survey elicited explanations for each program reduction. RESULTS: 200 (74%) of 269 surveys were returned. Eighty-four responders selected specific residencies to be reduced or eliminated. EM was selected 8 times, making EM the seventh most vulnerable residency to be targeted for reductions. The decision makers who selected proportional reductions chose to reduce across all residencies 32 times, among only the specialty residencies 129 times, and among only the primary care residencies 3 times. CONCLUSIONS: In the setting of anticipated residency cuts, favored proportional reductions in specialty residencies would likely affect EM training. However, most GME decision makers with an existing EM residency program do not consider the EM residency a top choice to be reduced or eliminated.     

The Homothorax homeoprotein activates the nuclear localization of another homeoprotein, extradenticle, and suppresses eye development in Drosophila

The Extradenticle (Exd) protein in Drosophila acts as a cofactor to homeotic proteins. Its nuclear localization is regulated. We report the cloning of the Drosophila homothorax (hth) gene, a homolog of the mouse Meis1 proto-oncogene that has a homeobox related to that of exd. Comparison with Meis1 finds two regions of high homology: a novel MH domain and the homeodomain. In imaginal discs, hth expression coincides with nuclear Exd. hth and exd also have virtually identical, mutant clonal phenotypes in adults. These results suggest that hth and exd function in the same pathway. We show that hth acts upstream of exd and is required and sufficient for Exd protein nuclear localization. We also show that hth and exd are both negative regulators of eye development; their mutant clones caused ectopic eye formation. Targeted expression of hth, but not of exd, in the eye disc abolished eye development completely. We suggest that hth acts with exd to delimit the eye field and prevent inappropriate eye development.