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.     

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.     

"My secret life": the emergence of one gay man''s authentic identity

Assessment of gestational age is one of the most important aims of ultrasonography in obstetrics. As pregnancy progresses, influences of factors which makes the growth of the fetuses different, cumulates. It has been revealed that the earlier in pregnancy ultrasound examination was performed, the better precision in the assessment of gestational age was obtained. The best precision is encountered when Crown-rump length is measured between 7-9 weeks of gestation. Until the end of the half of pregnancy, biparietal measurement allows for better assessment of gestational age than the date of the last menstrual period. As pregnancy progresses the variability of the measurements increases, reaching 7 week variation at term. The aim of this study is the assessment of fetal sacral length ultrasonographic measurements in the calculation of gestational age. The investigated group consists of 453 pregnant women between 16-41 weeks of physiological gestation.     

Shaker-1 mutations reveal roles for myosin VIIA in both development and function of cochlear hair cells

The mouse shaker-1 locus, Myo7a, encodes myosin VIIA and mutations in the orthologous gene in humans cause Usher syndrome type 1B or non-syndromic deafness. Myo7a is expressed very early in sensory hair cell development in the inner ear. We describe the effects of three mutations on cochlear hair cell development and function. In the Myo7a816SB and Myo7a6J mutants, stereocilia grow and form rows of graded heights as normal, but the bundles become progressively more disorganised. Most of these mutants show no gross electrophysiological responses, but some did show evidence of hair cell depolarisation despite the disorganisation of their bundles. In contrast, the original shaker-1 mutants, Myo7ash1, had normal early development of stereocilia bundles, but still showed abnormal cochlear responses. These findings suggest that myosin VIIA is required for normal stereocilia bundle organisation and has a role in the function of cochlear hair cells.     

A comparison between the Fick method and indirect calorimetry for determining oxygen consumption in patients with fulminant hepatic failure

OBJECTIVE: To compare the Fick method of determining oxygen consumption (VO2) with a gas exchange method in a group of patients in whom the cardiac output and mixed venous oxygen saturation values were consistently high. DESIGN: A prospective, observational study. SETTING: A ten-bed intensive therapy unit at a university teaching hospital. PATIENTS: Seventeen patients suffering from fulminant hepatic failure who required ventilatory support and invasive hemodynamic monitoring. All patients were sedated and paralyzed throughout the study period. INTERVENTIONS: None. MEASUREMENTS AND MAIN RESULTS: VO2 was determined simultaneously by indirect calorimetry and by the Fick method five or six times in each patient over a 5-hr period after resuscitation with fluids and, if clinically indicated, norepinephrine infusion. The agreement between the methods was poor (limits of agreement +19 to -101 mL/min/m2) and the Fick method consistently underestimated gas exchange measurements (mean bias 41 mL/min/m2). The bias varied widely, both between and within individual patients. The reproducibility of the Fick-derived VO2 was worse than the indirect calorimetry measurements, indicating that the dispersion of data attributable to measurement error was greater with the Fick method. CONCLUSIONS: Under clinical conditions, the agreement between Fick calculations and indirect calorimetry measurements of VO2 in hyperdynamic patients with fulminant hepatic failure was extremely poor. The reproducibility of Fick calculations was less than the reproducibility derived by gas exchange measurements because of the large measurement errors that may occur with the Fick method when the cardiac output is large and the arterial-venous oxygen content difference is small. Fick calculations systematically underestimate gas exchange measurements. The Fick method is inaccurate and unreliable when an estimation of VO2 is required in patients with this hemodynamic pattern.     

Hearing loss in community-dwelling older persons: national prevalence data and identification using simple questions

OBJECTIVE: To estimate the prevalence of hearing loss among community-dwelling older persons according to clinical criteria and to develop a brief self-report screening instrument to detect hearing loss. DESIGN: Survey. SETTING: National probability sample of noninstitutionalized older persons. PARTICIPANTS: A total of 2506 persons aged 55 to 74 who participated in the National Health and Nutrition Examination Survey. MAIN OUTCOME MEASURES: Hearing loss as defined by Ventry and Weinstein (VW) criteria and by the High Frequency Pure-Tone Average (HFPTA) scale. RESULTS: Hearing loss by VW criteria was present in 14.2% and by HFPTA criteria in 35.1% of those surveyed. The prevalence increased with advancing age and was higher among men and those with less education. A logistic regression model identified six independent factors for hearing loss by VW criteria: age > or = 70 years (adjusted odds-ratio (AOR) 2.7, 95% confidence interval (95% CI) 1.6, 4.4), male gender (AOR 3.0, 95% CI 1.9, 4.8), < or = 12th grade education (AOR 3.8, 95% CI 1.8, 7.7), having seen a doctor for deafness or hearing loss (AOR 8.9, 95% CI 5.3, 14.9), unable to hear a whisper across a room (AOR 3.2, 95% CI 2.0, 5.1), and unable to hear a normal voice across a room (AOR 6.2, 95% CI 2.6, 14.9). A clinical scale based on the logistic model had 80% sensitivity and 80% specificity in predicting hearing loss using VW criteria and 59% sensitivity and 88% specificity in predicting hearing loss using HFPTA criteria. CONCLUSIONS: Hearing loss, as defined by two clinical criteria, is common and can be screened for accurately using simple questions that assess sociodemographic and hearing-related characteristics.     

Influence of sympathectomy on the lateral hypothalamic lesion syndrome.

Two experiments with male Long-Evans rats examined sympathetic involvement in the lateral hypothalamic (LH) lesion syndrome. Ss were surgically or chemically sympathectomized and then given LH lesions. At 24 hrs postlesion, lesion-induced hyperglycemia but not hyperthermia was attenuated by splanchnicectomy and celiac ganglionectomy. Hyperthermia but not hyperglycemia was attenuated by adrenal demedullation, adrenalectomy, and daily neonatal guanethidine (50 mg/kg) treatment. Guanethidine-sympathectomized Ss also displayed lower basal temperatures, more perilesion chromatolysis, and more severe external symptoms than controls. No form of sympathectomy affected lesion-induced gastric pathology, plasma gastrin concentrations, or body weight loss, nor did any sympathectomy influence the recovery of ingestive behavior, daily food intake, the feeding response to 2-deoxy-dextro-glucose, or body weight maintenance in recovered LH-lesioned Ss. Results suggest that sympathetic hyperactivity contributed to some aspects of the acute LH syndrome: Hyperglycemia resulted from sympathetic outflow to the abdomen, whereas hyperthermia was determined by circulating catecholamines and extra-abdominal sympathetic innervation. Findings fail to support the hypothesis that chronic increases in sympathetic tone are responsible for the reduced food intake and body weight of the LH-lesioned Ss. (47 ref) (PsycINFO Database Record (c) 2010 APA, all rights reserved)     

The influence of surveillance methods on surgical wound infection rates in a tertiary care spinal surgery service

We studied 13 consecutive infants admitted to our Neonatal Intensive Care Unit over 37 months from 1 June 1994 to 30 June 1997, who were diagnosed with severe persistent pulmonary hypertension (PPHN) meeting extracorporeal membrane oxygenation (ECMO) criteria as defined by Bartlett and/or Short. They were managed with conservative ventilation strategy, with emphasis on the use of moderate ventilatory pressures whilst avoiding paralysis. Peak inspiratory pressure (PIP) on intermittent mandatory ventilation was adjusted according to adequate chest excursion. High PIP was avoided. Two main ventilatory techniques were used: 1) low ventilatory rate < or = 40/min, PIP 20 to 30 cmH2O, inspiratory time (IT) 0.5 seconds, positive end-expiratory pressure (PEEP) 5 cmH2O, and 2) high ventilatory rate 100/min, PEEP 0 cmH2O, IT 0.3 seconds. The aim was to keep preductal PaO2 > or = 50 mmHg. We did not sought to achieve alkalotic pH or low PaCO2. When PIP requirements exceeded 30 to 35 cmH2O, the use of an alternative rescue therapy such as pulmonary vasodilator, high frequency ventilation and/or surfactant were considered. Only 1 infant died of PPHN. Low mortality due to PPHN can be achieved using this strategy. There is a need for a randomised controlled trial to compare this strategy with other alternative treatment strategies.