Analysis of aerobic bacterial strains found in chronic rhinosinusitis using the polymerase chain reaction.

INTRODUCTION: Rhinosinusitis is a common disease affecting an estimated 14% of the population. Although there is general agreement in the literature regarding acute rhinosinusitis, chronic rhinosinusitis is not as well studied, and no consensus has been reached regarding the bacterial etiology. The goal of this study was to test chronic rhinosinusitis mucosal specimens using the polymerase chain reaction (PCR) for aerobic bacterial content and to compare the results with standard culture data. RESULTS: Routine culture samples grew 50% aerobic bacteria, whereas PCR detected 62% aerobic bacteria contamination. CONCLUSION: PCR detected more bacteria in mucosal samples than in standard culture, but standard culture of this mucosa reflects the general aerobic bacteria found in chronic rhinosinusitis, with no predominant species of aerobic bacteria. SIGNIFICANCE: The analysis of chronic rhinosinusitis mucosa with the PCR method should give a more accurate picture of the bacteria found in chronic rhinosinusitis so that proper therapy can be instituted.

The tension wire method: a simple, effective means of mandibular fixation.

OBJECTIVE: To investigate the use of a tension wire band secured to monocortical screws for open reduction and internal fixation of simple, displaced, and/or unstable mandibular fractures. DESIGN: Retrospective review with follow-up duration of at least 6 weeks. SETTING: Level I university trauma center. PATIENTS: Twenty-nine patients (27 males and 2 females), aged 7 to 46 years. Ten patients had unilateral fractures (1 patient had 3 separate unilateral fractures) and 19 had bilateral fractures; 34 of 50 fractures were displaced; 19 were open intraorally. The location of fractures and the number (number repaired) were as follows: symphysis or parasymphysis, 13 (10); body, 9 (8); angle, 18 (14); ramus, 6 (4); and subcondylar, 4 (0). Two fractures were each comminuted into 3 fragments, and 1 patient had unilateral parasymphysial, body, and ramus fractures. INTERVENTION: Intermaxillary fixation was done in all patients except 1 child. Intraoral approaches were used exclusively. A pair of monocortical 2.0-mm screws were placed perpendicular to fracture lines, with 24-gauge wire loops passed around the screws and tightened to bring the fracture into reduction and provide stable fixation. A percutaneous trocar system was used to insert screws at the body, angle, and ramus sites. RESULTS: There were no instances of infection, malunion, or malocclusion in the 33 fractures repaired with this technique. A typical intermaxillary fixation with open reduction and internal fixation of a posterior fracture was done in less than 2 hours. CONCLUSION: Open reduction and internal fixation of simple mandibular fractures with tension wire bands around monocortical screws is a simple, quick, and effective technique.

Accuracy of clinical evaluation in pediatric obstructive sleep apnea.

Eighty-two children underwent polysomnography (PSG) for symptoms suggestive of obstructive sleep apnea (OSA). Symptoms reported included snoring, witnessed apneic episodes, daytime somnolence, mouth breathing, and enuresis. Tonsillar size, nasal airway patency, and percentile weight were recorded. OSA was diagnosed on PSG when obstructive events were noted and apnea + hypopnea index was five or more per hour. The overall predictive accuracy of clinical suspicion of OSA was 25 (30%) of 82. Predictive accuracies (as a percentage of those with symptoms/signs who have OSA) and prevalences (as a percentage of those with OSA who have the symptom/sign), respectively, were for moderate snoring 29% (12 of 41), 48%; loud snoring 31% (11 of 35), 44%; witnessed apneas 32% (22 of 69), 88%; enuresis 46% (11 of 24), 44%; 2+ tonsillar size 37% (21 of 57), 84%; 3+ tonsillar size 33% (3 of 9), 12%; 90th percentile weight or greater 26% (7 of 27), 28%; 10th percentile weight or less 33% (5 of 15), 20%. Multiple regression analysis did not reveal a significant association between clinical parameters and the presence of OSA as defined by PSG.

Isolation of a carboxyphosphate intermediate and the locus of acetyl-CoA action in the pyruvate carboxylase reaction.

When chicken liver pyruvate carboxylase was incubated with either H14CO3- or gamma-[32P]ATP, a labeled carboxyphospho-enzyme intermediate could be isolated. The complex was catalytically competent, as determined by its subsequent ability to transfer either 14CO2 to pyruvate or 32P to ADP. While the carboxyphospho-enzyme complex was inherently unstable and the stoichiometry of the transfer was variable depending on experimental conditions, both the [14C]carboxyphospho-enzyme and the carboxy[32P]phospho-enzyme had similar half-lives. Acetyl-CoA was shown to be involved in the conversion of the carboxyphospho-enzyme complex to the more stable carboxybiotin-enzyme species, which was consistent with the effects of acetyl-CoA on isotope exchange reactions involving ATP. We were unable to detect the formation of a phosphorylated biotin derivative during the ATP cleavage reaction. In the presence of K+ and at pH 9.5, the acetyl-CoA-independent activity of chicken liver pyruvate carboxylase approached 2% of the acetyl-CoA-stimulated rate, which represents a 30-fold increase on previously reported activity for this enzyme.

Further studies on the localization of the reactive lysyl residue of pyruvate carboxylase.

We have shown the increase in the acetyl-CoA-independent activity of sheep liver pyruvate carboxylase following trinitrophenylation of a specific lysine residue (designated Lys-A) to be the result of a large stimulation of the first partial reaction and a slight stimulation of the second partial reaction catalysed by this enzyme. Like acetyl-CoA, the activators adenosine 3',5'-bisphosphate and CoA did not stimulate the catalytic activity of the trinitrophenylated enzyme in either the overall reaction or the first partial reaction. Conversely, trinitrophenylation had no effect on activation of the overall reaction and the second partial reaction by acetyl-phosphopantetheine. Protection experiments demonstrated that the presence of both acetyl-CoA and adenosine 3',5'-bisphosphate decreased the rate of loss of activity during exposure of sheep liver pyruvate carboxylase to trinitrobenzenesulphonic acid (TNBS), whereas acetyl-phosphopantetheine did not. 5'-AMP and acetyl-dephospho-CoA did not protect the enzyme against loss of activity, whereas the presence of adenosine 2',5'-bisphosphate only slightly decreased the rate of modification. This suggests that Lys-A interacts with the adenosine nucleotide portion of the acetyl-CoA molecule, specifically the 3'-phosphate moiety. Acetyl-CoA and adenosine 3',5'-bisphosphate were shown to protect pyruvate carboxylase from Saccharomyces cerevisiae against inhibition by TNBS. A [14C]acetyl-CoA-binding assay demonstrated that modification of Lys-A inhibits the binding of acetyl-CoA to S. cerevisiae pyruvate carboxylase, indicating that Lys-A is at or near the acetyl-CoA-binding site.

Studies on dilution inactivation of sheep liver pyruvate carboxylase.

When sheep liver pyruvate carboxylase was diluted below 4 EU/ml, it underwent inactivation involving two kinetically distinct processes, i.e., a rapid initial burst followed by a slower second phase. The catalytic activity of the diluted enzyme eventually approached zero, suggesting the occurrence of an irreversible process. Analysis of the quaternary structure of the enzyme by gel filtration chromatography and electron microscopy showed that most of the enzyme molecules occur as tetramers at high enzyme concentrations. However, dilution of the enzyme below 4 EU/ml led to the appearance of dimers and monomers which were essentially inactive under the conditions of the assay system used. The presence of acetyl-CoA during dilution prevented inactivation from occurring and preserved the tetrameric structure. When added after dilution, acetyl-CoA prevented further inactivation from occurring but did not reactivate the enzyme. However, acetyl-CoA did cause a relatively rapid reassociation of the inactive monomers and dimers to form inactive tetramers.

Inactivation of chicken liver pyruvate carboxylase by 1,10-phenanthroline.

Inactivation of chicken liver pyruvate carboxylase by the chelating agent 1,10-phenanthroline follows pseudo-first-order kinetics. The hyperbolic dependence of the apparent first-order rate constant on 1,10-phenanthroline concentration is consistent with a two-step inactivation mechanism, in which 1,10-phenanthroline binds firstly to the enzyme, and secondly to the enzyme-bound Mn(II) ion. Binding of 1,10-phenanthroline to pyruvate carboxylase results in complete loss of ATP/Pi exchange activity, but only a 61% decrease in pyruvate/oxaloacetate exchange activity. The rate of inactivation is greater at low enzyme concentrations, implying that binding of 1,10-phenanthroline to monomers and dimers is preferred relative to that of tetramers. Furthermore, in the presence of acetyl-CoA, which stabilizes the tetrameric structure, no dependence of inactivation on enzyme concentration is observed. As monitored by gel-permeation liquid chromatography, formation of the enzyme-Mn(II)-phenanthroline complex results in loss of the tetrameric structure of the enzyme. From atomic-absorption measurements, inactivation by 1,10-phenanthroline also causes some loss of Mn(II) from the enzyme. It is concluded that the Mn(II) atom does not participate directly in the reaction mechanism, but may play a structural role essential to the integrity of the enzyme's tetrameric structure.

Pyruvate carboxylase: mechanisms of the partial reactions.

Data from isotopic exchange studies and from experiments with 32P- and 14C-labeled enzyme-bound intermediates support the following description of the first partial reaction: (Formula: see text). From studies of the transfer of the carboxyl-group from ENZ-biotin-CO2- to pyruvate or its analogues we propose that binding of the acceptor substrate induces the translation of carboxybiotin from the first to the second partial reaction site. The studies on the translocation of carboxybiotin can be summarized in the following reaction scheme: (Formula: see text). Where k+3 less than k+1, k-1, k+2 and k-2. Thus, the rate-limited step is governed by k+3 which represents the movement of carboxybiotin from the first subsite to the second.

The carboxybiotin complex of pyruvate carboxylase. A kinetic analysis of the effects of Mg2+ ions on its stability and on its reaction with pyruvate.

The enzyme-[14C] carboxybiotin complex of sheep liver pyruvate carboxylase was isolated and the reaction between this and pyruvate was studied by using the quenched-flow rapid-reaction technique. At 0.5 degrees C the reaction was 80% complete within 180 ms. The reaction was monophasic and obeyed pseudo-first-order kinetics. Increasing concentrations of Mg2+ caused a decrease in the magnitude of the observed pseudo-first-order rate constant. Throughout the carboxylation of pyruvate, the rate-limiting step of the reaction occurred after the dissociation of carboxybiotin from the first sub-site, whereas in the slow phase of the reaction with 2-oxobutyrate this dissociation is the rate-limiting step. It is possible, from the reaction scheme proposed, that the inhibition of overall enzymic activity by high concentrations of Mg2+ could be caused by the transfer of the carboxy group from biotin to pyruvate becoming rate-limiting. The efficacy of a substrate as a signal for the movement of carboxybiotin from the first sub-site is reflected by the amount that the effective affinity of the enzyme- carboxybiotin complex for Mg2+ is lowered. In the presence of the substrates tested, the affinities of the carboxybiotin complex can be arranged in order of increasing magnitude, i.e.: (formula; see text). The kinetics of the decay of the enzyme-[14C] carboxybiotin complex at 0 degree C in the absence of substrates are similar to the reaction with pyruvate except that the carboxybiotin is also unstable in the first sub-site, to some degree. This similarity allows for the proposal of a general scheme for the decarboxylation of the enzyme- carboxybiotin complex in the presence or in the absence of substrates.

A mechanism for the transfer of the carboxyl-group from 1'-N-carboxybiotin to acceptor substrates by biotin-containing enzymes.

Previous proposals for the mechanism by which biotin-dependent enzymes catalyse the transfer of the carboxyl group from 1'-N-carboxybiotin to acceptor molecules do not appear to be consistent with all of the experimental observations now available. We propose a multi-step mechanism in which (a) substrate and then carboxybiotin bind at the second partial reaction site, (b) a base positioned adjacent to the 3'-N of the carboxybiotin abstracts a proton from the 3'-N and (c) the resulting enolate ion and the acceptor substrate undergo a concerted reaction resulting in carboxyl-group transfer.

Localisation of the active site of pyruvate carboxylase by electron microscopic examination of avidin-enzyme complexes.

Negatively stained enzyme-avidin complexes, seen at different stages of the titration of the biotin-binding sites on avidin with chicken liver pyruvate carboxylase, have been studied using electron microscopy. Formation of linear, unbranched polymers of the enzyme-avidin complex is seen to occur when the ratio of avidin to enzyme is between 2:1 and 1:2; beyond these limits only single enzyme tetramers are visible. The single avidin molecules observed seem to have a cuboid structure. The orientation and dimensions of the enzyme tetramers within the polymers indicate that the tetrahedron-like structure, observed in the single molecules, has been preserved. From the structure of the polymers and the observation of single enzyme-avidin complexes, we deduce that the biotin groups on the enzyme are located on the external faces of each subunit close to, and probably within 3 nm of, the intersubunit junction.

Factors that influence the translocation of the N-carboxybiotin moiety between the two sub-sites of pyruvate carboxylase.

The active site of pyruvate carboxylase, like those of all biotin-dependent carboxylases, is believed to consist of two spatially distinct sub-sites with biotin acting as a mobile carboxy-group carrier oscillating between the two sub-sites. Some of the factors that influence the location and rate of movement of the N-carboxybiotin were studied. The rate of carboxylation of the alternative substrate, 2-oxobutyrate, was measured at 0 degrees C in an assay system where the isolated enzyme--[14C]carboxybiotin was the carboxy-group donor. The results are consistent with the hypothesis that the location of the carboxybiotin in the active site is determined by the presence of Mg2+, acetyl-CoA and the oxo acid substrate. The presence of Mg2+ favours the holding of the complex at the first sub-site, whereas alpha-oxo acids induce the complex to move to the second sub-site. At low concentrations pyruvate induces this movement but does not efficiently act as a carboxy-group acceptor; hydroxypyruvate, glyoxylate and oxamate, though not carboxylated, still induce the movement. The allosteric activator acetyl-CoA exerts only a slight stimulation on the rate of translocation to the second sub-site, and this stimulation arises from an increase in the dissociation constant for Mg2+.

Further electron microscope studies on pyruvate carboxylase.

Using negative staining and electron microscopic tilting techniques in conjunction with modelling experiments, the fine structure of chicken, sheep and rat pyruvate carboxylases has been studied. The overall configuration appears to be a tetrahedron-like structure consisting of two pairs of subunits in two different planes orthogonal to each other with the opposing pairs of subunits interacting with each other on their convex surfaces. The predominant form of the enzyme particles mounted and stained in the presence of acetyl-coenzyme A consisted of a compact, triangular outline enclosing three readily visible intensity maxima. When samples were mounted in the absence of acetyl-coenzyme A the molecules were more 'open' predominantly rhomboid structures. From tilting experiments it is concluded that the rhomboid images found in the absence of acetyl-coenzyme A represent partly or wholly flattened forms of the tetrahedron-like molecule. A feature of the enzyme when mounted in the absence of acetyl-coenzyme A was the existence of a 'cleft' along the longitudinal midline of each subunit, suggesting that the subunits may consist of two distinct domains.

The atypical velocity response by pyruvate carboxylase to increasing concentrations of acetyl-coenzyme A.

An investigation was made of the interaction of pyruvate carboxylase with its allosteric effector, acetyl-CoA, and the velocity profile of the deacylation of acetyl-CoA as a function of acetyl-CoA concentration indicated that this ligand does not bind to this enzyme in a positive homotropic co-operative manner. An examination was therefore made of the factors that contribute to the sigmoidicity of the rate curves obtained for pyruvate carboxylation with various concentrations of acetyl-CoA. Hill coefficients for acetyl-CoA obtained with both sheep and chicken liver pyruvate carboxylases were found to be dependent on the fixed pyruvate concentration used in the assay solution. Thus, by varying the acetyl-CoA concentration, the degree of saturation of the enzyme by pyruvate was also changed. A further consequence of non-saturating concentrations of pyruvate was that the non-productive hydrolysis of the enzyme- carboxybiotin complex increased, resulting in an under-estimate of the reaction velocity measured by oxaloacetate formation. Another factor contributing to the sigmoidicity is that, at non-saturating concentrations of acetyl-CoA, the enzyme undergoes inactivation upon dilution to low protein concentrations, again resulting in an under-estimate of the reaction velocity. Under conditions where none of the above factors was operating and the only effect of varying acetyl-CoA concentrations was to alter the proportion of the enzyme catalysing the carboxylation reaction at acetyl-CoA-dependent and -independent rates, the sigmoidicity of the acetyl-CoA velocity profile was completely eliminated.