Biomedical subjects

B E Smith

Publications and source records attributed to B E Smith.

At least 37 records · Page 2Linked to original sources

PubMed1998

Assessment of clinical performance during simulated crises using both technical and behavioral ratings.

BACKGROUND: Techniques are needed to assess anesthesiologists' performance when responding to critical events. Patient simulators allow presentation of similar crisis situations to different clinicians. This study evaluated ratings of performance, and the interrater variability of the ratings, made by multiple independent observers viewing videotapes of simulated crises. METHODS: Raters scored the videotapes of 14 different teams that were managing two scenarios: malignant hyperthermia (MH) and cardiac arrest. Technical performance and crisis management behaviors were rated. Technical ratings could range from 0.0 to 1.0 based on scenario-specific checklists of appropriate actions. Ratings of 12 crisis management behaviors were made using a five-point ordinal scale. Several statistical assessments of interrater variability were applied. RESULTS: Technical ratings were high for most teams in both scenarios (0.78 +/- 0.08 for MH, 0.83 +/- 0.06 for cardiac arrest). Ratings of crisis management behavior varied, with some teams rated as minimally acceptable or poor (28% for MH, 14% for cardiac arrest). The agreement between raters was fair to excellent, depending on the item rated and the statistical test used. CONCLUSIONS: Both technical and behavioral performance can be assessed from videotapes of simulations. The behavioral rating system can be improved; one particular difficulty was aggregating a single rating for a behavior that fluctuated over time. These performance assessment tools might be useful for educational research or for tracking a resident's progress. The rating system needs more refinement before it can be used to assess clinical competence for residency graduation or board certification.

Anesthesiology↗

PubMed1998

Spinal anesthesia for cesarean section. 1963.

Safety in spinal anesthesia for cesarean section is achieved by strict adherence to the cardinal principles of proper evaluation and selection of patients, the use of prophylactic vasopressors, preanesthetic establishment of a reliable intravenous channel, small doses of the local anesthetic, close monitoring and maintenance of systolic blood pressure above 100 mm. Hg, and avoidance of ergot compounds in the presence of vasopressors.

Anesthesia, Obstetrical↗

PubMed1997

Structures of a blue-copper nitrite reductase and its substrate-bound complex.

Copper-containing nitrite reductases (NiR's) have been conveniently subdivided into blue and green NiR's which are thought to be redox partners of azurins and pseudo-azurins, respectively. Crystal structures of two green NiR's have recently been determined. Alcaligenes xylosoxidans has been shown to have a blue-copper nitrite reductase (AxNiR) and two azurins with 67% homology both of which donate electrons to it effectively. The first crystal structure of a blue NiR (AxNiR) in its oxidized and nitrite-bound forms, with particular emphasis to the Cu sites, is presented. The Cu-Smet distance is the same as those in the green NiR's. Thus, the length of this interaction is unlikely to be responsible for differences in colour. Crystallographic data presented here taken together with structural data of other single Cu type-1 proteins and their mutants suggest that the displacement of Cu from the strong ligand plane is perhaps the cause for the differences in colour observed for otherwise 'classical' blue Cu centre. Nitrite is observed binding to the catalytic Cu in a bidentate fashion displacing the water molecule, offering a neat rationalization for the XAFS observation that the type-2 Cu-ligand distances increase on nitrite binding as a result of increased coordination. These results are discussed in terms of enzyme mechanism.

Journal Article↗

PubMed1997

pH-dependence for binding a single nitrite ion to each type-2 copper centre in the copper-containing nitrite reductase of Alcaligenes xylosoxidans.

The first quantitative characterization of the interaction of NO2(-) with the Cu-containing dissimilatory nitrite reductase (NiR) of Alcaligenes xylosoxidans using steady-state kinetics, equilibrium gel filtration and EPR spectroscopy is described. Each molecule of this protein consists of three equivalent subunits, each containing a type-1 Cu atom and also a type-2 Cu atom at each subunit interface. Enzyme activity increased in a biphasic manner with decreasing pH, having an optimum at pH 5.2 and a plateau between pH 6.1 and 5.8. Equilibrium gel filtration showed that binding of NO2(-) to the oxidized NiR was also pH-dependent. At pH 7.5, no binding was detectable, but binding was detectable at lower pH values. At pH 5.2, the concentration-dependence for binding of NO2(-) to the enzyme showed that approx. 4.1 NO2(-) ions bound per trimeric NiR molecule. Unexpectedly, NiR deficient in type-2 Cu centres bound 1.3 NO2(-) ions per trimer. When corrected for this binding, a value of 3 NO2(-) ions bound per trimer of NiR, equivalent to the type-2 Cu content. The NO2(-)-induced changes in the EPR parameters of the type-2 Cu centre of the oxidized enzyme showed a similar pH-dependence to that of the activity. Binding constants for NO2(-) at a single type of site, after allowing for the non-specifically bound NO2(-), were 350+/-35 microM (mean+/-S.E.M.) at pH 7.5 and <30 microM at pH 5.2. The apparent Km for NO2(-) with saturating concentrations of dithionite as reductant was 35 microM at pH 7.5, which is 10-fold tighter than for the oxidized enzyme, and is compatible with an ordered mechanism in which the enzyme is reduced before NO2(-) binds.

Alcaligenes↗

PubMed1997

Covalent modification of nitrogenase MoFe protein by ADP.

MgADP- reacted with the nitrogenase molybdenum-iron (MoFe) protein of Klebsiella pneumoniae (Kp1) over a period of 2 h to yield a stable, catalytically active conjugate. The isolated protein exhibited a new, broad 31P NMR resonance at -1 p.p.m. lacking phosphorus J coupling. The adenine ring of [8-14C]ADP remained associated with the conjugate. A covalently bound nucleotide was identified as AMP by NMR and TLC. Extended dialysis of Kp1 against MgADP- resulted in further AMP binding at the protein surface. ADP was initially bound tightly to Kp1 at a site distinct from the AMP sites. ATP did not replace ADP. The time course of the formation of the Kp1-AMP was altered by the nitrogenase iron protein (Kp2) and was dependent on redox potential. Kp1-AMP was stable to concentration and oxidation with ferricyanide ion at -350 mV. Slow hydrolysis of Kp1-AMP over a period of 6 h yielded AMP and unaltered Kp1. The adenine ring of ADP exchanged with adenine of MgATP2- during reductant-limited turnover of nitrogenase under N2, indicating reversibility of ATP hydrolysis at 15 degrees C. [32P]Pi exchanged with the terminal phosphate group of both ADP and ATP on incubation with Kp1. 32P exchange and the catalytic activity of Kp1 were inhibited by a 20-fold molar excess of the lysine-modifying reagent, o-phthalaldehyde (OPT). Preincubation with MgADP- protected against OPT inactivation. Two potentially reactive lysine residues on the alpha chain of the MoFe protein near a putative hydrophobic docking site for the nitrogenase Fe protein are proposed as sites of OPT and nucleotide binding. Azotobacter vinelandii MoFe protein (Av1) also formed an AMP adduct but Kp2 did not. Catalase did not interact with ADP. The reactions of the nitrogenase MoFe protein with adenine nucleotides have no counterpart in known protein-nucleotide interactions.

Adenosine Diphosphate↗

PubMed1997

The first glimpse of a complex of nitrogenase component proteins by solution X-ray scattering: conformation of the electron transfer transition state complex of Klebsiella pneumoniae nitrogenase.

An essential feature of the mechanism of nitrogenase, the enzyme responsible for biological nitrogen fixation, is the formation of a transient electron transfer complex between the MoFe protein containing the active site at which N2 is reduced, and the Fe protein, which functions as a specific electron donor to the MoFe protein. We have obtained high quality solution X-ray scattering data using synchrotron X-rays of a stable putative electron transfer complex, (MoFe-protein)(Fe-protein.ADP.AIF4)2, of Klebsiella pneumoniae and used the model-independent approach based on the multipole expansion method to provide a stable and unique shape restoration at approximately 15 A resolution. The biological significance of this first molecular structure of a nitrogenase complex is discussed.

Azotobacter vinelandii↗

PubMed1997

Crystallization and preliminary X-ray studies of nitrogenase component 1 (the MoFe protein) from Klebsiella pneumoniae.

Two crystal forms of component 1 (the MoFe protein) of nitrogenase from Klebsiella pneumoniae have been isolated and characterized. The triclinic form has cell dimensions a = 76.0, b = 109.6, c = 144.6 A, alpha = 80.3, beta = 74.9 and gamma = 69.6 degrees, diffracts to around 3.0 A and has two molecules in the asymmetric unit. The monoclinic form belongs to space group P2(1) with a = 76.6, b = 127.8, c = 109.1 A and beta = 104.6 degrees (frozen at 100 K), diffracts to 1.5 A and has one molecule in the asymmetric unit. At this resolution the outstanding questions concerning the structure and the operation of the enzyme, in particular the linkage between the Fe(4)S(4) units in the P clusters, the true geometry of the apparently trigonal Fe atoms in the FeMoco and the reduction site itself, should be answerable.

Journal Article↗

PubMed1997

Evaluation of a medical diagnosis system using simulator test scenarios.

This paper describes an informal but systematic method for how to test and verify a knowledge-based system in a large open-ended medical target domain. The system used is Guardian, an intelligent system for monitoring and diagnosis of post-cardiac surgery patients in an intensive-care unit. The knowledge base is tested and verified by running the system on a series of realistic test scenarios, both with an embedded simulator and with an external simulation system. The same scenarios are presented to human test subjects, making it possible to compare and analyze the performance of the knowledge-based system with that of human physicians. The use of simulators instead of clinical data also means that it is possible to test crucial scenarios which occur seldom in medical practice. Our results show that a system like Guardian might indeed be useful in medical care.

Artificial Intelligence↗

PubMed1997

Effect of septal perforations on measures of nasal resistance.

OBJECTIVE: The purpose of this investigation was to determine the effect of septal perforations on posterior and anterior rhinomanometric measures of nasal resistance in an analog model. DESIGN: The data were analyzed using a repeated-measures ANOVA. Nasal resistance was the dependent variable, while type of rhinomanometry, septal perforation size, and position of resistance (proximal vs. distal) were nominal scale independent variables. PARTICIPANTS: The analog model used in this study was similar to that described by Warren and Devereux (1966), except that the nasal cavities of the model were modified to create septal perforations. OUTCOME MEASURES: The main dependent measure was nasal resistance. RESULTS: An Important finding of this investigation was that septal perforations resulted in large differences (> 3 cm H2O/L/sec) between posterior and anterior nasal resistance values in the bilateral proximal resistor condition. CONCLUSIONS: Anterior rhinomanometry may underestimate true nasal resistance when a septal perforation is present, because the septal perforation prevents accurate measurement of nasopharyngeal pressure. Posterior rhinomanometric measures should accurately reflect nasal resistance despite septal perforations, because the perforation does not invalidate the estimate of nasopharyngeal pressure.

Adult↗

PubMed1996

Isolation and characterization of nitrogenase MoFe protein from the mutant strain pHK17 of Klebsiella pneumoniae in which the two bridging cysteine residues of the P-clusters are replaced by the non-coordinating amino acid alanine.

Nitrogenase MoFe protein (Kp1) from the mutant strain pHK17 or Klebsiella pneumoniae has been purified to give three catalytically active fractions. In this mutant, each of the two bridging cysteine ligands to the P-clusters, alpha-Cys-89 and beta-Cys-94, has been replaced by a non-coordinating residue, alanine. SDS/PAGE and earlier native gels showed that the three fractions retained the normal alpha 2 beta 2 tetrameric form of wild-type Kp1; therefore we conclude that in each of the fractions the subunits are folded differently, thus resulting in different surface charges and allowing separation of the fractions on ion-exchange chronatography. Earlier EPR and magnetic CD data had shown that the mutant fractions contain P-clusters, and thus the mutated residues are not as essential for maintaining the integrity of the P-clusters as they appear from the X-ray structure. The specific activity of each of the three fractions was less than that of wild-type Kp1, the most active fraction having only 50% of wild-type activity. No change in substrate specificity or in the relative distribution of electrons to various substrates was found. The relationship between ATP hydrolysis and substrate-reducing activity, the EPR spectra of the S = 3/2 spin state of the iron-molybdenum cofactor (FeMoco) and the pH profile of acetylene-reduction activities of the three fractions did not differ significantly from those exhibited by wild-type Kp1. The specific activities of the three mutant fractions and of wild-type Kp1 were linearly proportional to the intensity of the S = 3/2 EPR signal from the FeMoco centres. This implies that those molecules of the three mutant fractions and the wild-type protein that contain EPR-active FeMoco are fully active, i.e. that the Cys to Ala substitution of the P-cluster ligands does not affect the specific activity of the protein. This in turn implies that the P-clusters are not directly associated with the rate-limiting step in enzyme turnover. We conclude that the lower specific activities of the mutant fractions are observed because the fractions are mixtures of species containing a full complement of FeMoco and P-clusters and species lacking some or all of these clusters. On the basis of the Mo contents and EPR spectroscopy of the mutant fractions, we propose that the loss of the P-clusters causes (i) the physical loss or inhibition of binding of some FeMoco; (ii) the EPR and catalytic inactivation of some FeMoco; and/or (iii) the incorporation of a FeMoco-like species into the FeMoco site of the mutant molecules.

Adenosine Triphosphate↗

PubMed1996

Long-term follow-up of tracheoesophageal puncture results.

Since its introduction by Blom and Singer in 1980, tracheoesophageal puncture with a voice prosthesis has become the most frequently recommended choice for speech rehabilitation of total laryngectomies. Many studies have reviewed the initial speech acquisition success rates following tracheoesophageal puncture; however, long-term follow-up in these initial successes has been lacking. In addition, factors predictive of long-term success with tracheoesophageal speech have not been defined. Over a 10-year period, we retrospectively reviewed all total laryngectomy patients, including those who have undergone primary or secondary tracheoesophageal puncture, at the University of Illinois Hospital and Clinics and the Westside Veterans Administration Hospitals. Survival in the total laryngectomy cohort of 202 patients ranged from 35% to 50%. Forty of these patients underwent tracheoesophageal puncture, in whom survival was 75%. Short-term success with tracheoesophageal speech was approximately 70% for our patients, while long-term success was achieved in 66%. Despite low socioeconomic status and relatively high alcoholism rates, successful maintenance of tracheoesophageal speech was achieved in the majority of cases. Tracheoesophageal speech should therefore be considered as a primary method of vocal rehabilitation in all patients undergoing total laryngectomy.

Adult↗

PubMed1996

Pressure-flow differences in performance during production of the CV syllables /pi/ and /pa/.

Inconsistencies in velopharyngeal function were observed to have been reported in pressure-flow reports. This article introduces our findings from a sample of pressure-flow records and discusses possible implications for management. A retrospective review of the pressure-flow records for 51 patients was completed. All patients had been evaluated at the Craniofacial Center, University of Illinois at Chicago. Records were selected based on the following criteria: patients were referred for evaluation of velopharyngeal function and had no secondary surgeries on the velopharyngeal mechanism, no fistulae, no neurologic disorders, and no compensatory articulations. At the time of testing, patients ranged in age from 4 to 38 years with most being under 18 years of age. Velopharyngeal orifice areas were obtained using the pressure-flow technique during repeated CV syllables, including /pi/ and /pa/. Our results show that 8 of 51 patients exhibited a specific type of inconsistent velopharyngeal function (i.e., they exhibited velopharyngeal closing during /pa/ repetitions, but had openings during /pi/ repetitions). The reverse finding was not observed among any of the 51 patients. We concluded that the velopharyngeal incompetence observed during utterances involving /i/ may have been due to the downward pull on the palate of the palatoglossus muscle, which could not be counteracted by the already maximized levator activity in borderline patients.

Adolescent↗

PubMed1996

Human SY5Y neuroblastoma cell interactions with laminin isoforms: neurite outgrowth on laminin-5 is mediated by integrin alpha 3 beta 1.

Laminin (Ln) isoforms may play important roles in neuronal development, particularly axon guidance, but neural receptors mediating interactions with Ln are not entirely understood. In this paper, we have compared the adhesive and process outgrowth activities of a human neuroblastoma cell line SY5Y on various laminin isoforms. Cell adhesion and process outgrowth were examined on murine Ln-1 (Englebreth-Holm-Swarm sarcoma laminin), human placental Ln-1 (human Ln-1[p]), human Ln-2 (merosin), human Ln-5 (kalinin/epiligrin/nicein), and human foreskin keratinocyte extracellular matrix extract (human HFK-ECM). Ln-5 was shown to evoke process outgrowth in amounts comparable to other Ln isoforms. Antibody perturbation experiments showed that adhesion and process outgrowth on murine Ln-1 was primarily mediated by the integrin alpha 1 beta 1, whereas adhesion and outgrowth on human Ln-5 and human HFK-ECM were mediated by alpha 3 beta 1. Adhesion to human Ln-1(p) and Ln-2 was not blocked by addition of anti-alpha 1 or anti-alpha 3 antibodies alone, but adhesion was partially perturbed when these antibodies were added in combination. Process outgrowth on human Ln-1(p) was blocked when either anti-alpha 3 or anti-beta 1 antibodies were added, indicating that alpha 3 beta 1 is the primary integrin heterodimer responsible for process extension on this substrate. These results demonstrate that Ln-5 and other Ln isoforms support comparable levels of adhesion and process outgrowth, but different integrin heterodimers, alone and in combination, are used by SY5Y cells to mediate responses.

Animals↗

PubMed1996

Determinants of prolonged oral cecal transit time during late phase pregnancy.

We examined the course of delayed oral cecal transit time (OCTT) and the impact of different variables in late pregnancy. Thirty-three women in late trimester were evaluated and 29 were re-tested post partum. Results were compared with 17 controls. A breath hydrogen test was used to measure OCTT. The impact of 3 demographic and 5 biochemical parameters was evaluated. Oral cecal transit time (99.2 +/- 7.8 min) during pregnancy was significantly prolonged compared both with controls (63.5 +/- 8.7 min) (p = 0.0042) and post partum (68.5 +/- 6.4 min) (p < 0.002). However, 40% during pregnancy had a normal OCTT. Post partum, 8 (29.6%) continued to have delayed OCTT. Serum free T4 levels were significantly lower than in controls (p < 0.0001) and post partum (p < 0.001), but did not correlate with OCTT. Gravidity rank correlated with OCTT (p = 0.022). By multivariate analysis, only 17% of the observed variability during pregnancy could be explained by these parameters. In controls, a formula including progesterone and gastrin levels was significant in explaining 54.7% of variability. Application of both formulae to post-partum returnees overpredicted OCTT; however, the one from pregnancy was more accurate. We conclude that in pregnancy multiple factors account for delayed OCTT and these may be different from controls and post partum. Pregnancy may still influence OCTT post partum.

Adult↗

PubMed1995

Structure of a new azurin from the denitrifying bacterium Alcaligenes xylosoxidans at high resolution.

It has been reported previously that Alcaligenes xylosoxidans (NC1MB 11015) grown under denitrifying conditions produces two azurins instead of the single previously identified azurin [Dodd, Hasnain, Hunter, Abraham, Debenham, Kanzler, Eldridge, Eady, Ambler & Smith (1995). Biochemistry. In the press]. The new azurin, called azurin II, has been crystallized as blue elongated rectangular prisms with the tetragonal space group P4(1)22 and unit-cell parameters a = b = 52.65, c = 100.63 A. X-ray crystallographic data extending to 1.9 A resolution were collected by the Weissenberg method using 200 x 400 mm image plates and synchrotron X-rays of wavelength 0.97 A. The three-dimensional structure of azurin II has been solved by the molecular-replacement method using the structure of azurin from Alcaligenes denitrificans NCTC 8582 with which this new azurin shows a close homology. The quality of the initial map was sufficient to predict a number of sequence differences. The model is currently refined to an R-factor of 18.8% with X-ray data between 8.5 and 1.9 A. The final model of 961 protein atoms, one Cu atom and 50 water molecules has r.m.s. deviations from ideality of 0.009 A for bond lengths and 1.7 degrees for bond angles. The overall structure is similar to that of the azurin from A. denitrificans NCTC 8582. It has a beta-barrel structure with the Cu atom located near the top end of the molecule. The Cu atom is coordinated to Ndelta of His46 and His117 at 2.02 A and to Sgamma of Cys112 at 2.12 A, while the carbonyl O atom of Gly45 and Sdelta atom of Met121 provide the additional interactions at 2.75 and 3.26 A, respectively.

Journal Article↗

PubMed1995

Evidence for two distinct azurins in Alcaligenes xylosoxidans (NCIMB 11015): potential electron donors to nitrite reductase.

We have isolated two type 1 copper-containing proteins (M(r) approximately 13K) from Alcaligenes xylosoxidans (NCIMB 11015) grown under denitrifying conditions. Amino acid sequence analysis of these two proteins shows one to be the previously identified azurin (Ambler, 1971), which we shall call azurin I, and the other to be a related, but previously undescribed, blue copper protein which we show to also be an azurin and propose to call azurin II. Thus, NCIMB 11015 becomes the second system where two distinct azurins are found, the other being Methylomonas J (Ambler & Tobari, 1989). On isoelectric focusing, azurin I migrates very similarly to the previously identified azurin from this organism while azurin II migrates similarly to azurin purified from Alcaligenes denitrificans NCTC 8582. The sequence of azurin II is 33% different than the azurin I sequence but is only 11% different than the azurin from Alcaligenes denitrificans NCTC 8582. Optical spectra for the two proteins are very similar with epsilon mM values of 6.27 and 5.73 mM-1 cm-1 for azurin I and II, respectively, at lambda max approximately 620 nm. The 291 nm shoulder normally ascribed to the hydrophobic nature of tryptophan 48 is clearly observed in azurin I but is missing in azurin II. Amino acid analysis confirms that this tryptophan is missing in azurin II. Azurin I and azurin II show essentially the same redox potential of 305 +/- 10 mV at pH 7.5 and are equally effective electron donors to the purified dissimilatory nitrite reductase of Alc. xylosoxidans in vitro.(ABSTRACT TRUNCATED AT 250 WORDS)

Alcaligenes↗

PubMed1995

Lithium in the mating response and cell cycle of Saccharomyces cerevisiae.

Studies with Li+, an inhibitor of phosphoinositide metabolism, demonstrated that an early response of Saccharomyces cerevisiae to a-factor pheromone was negatively affected by this cation. This was monitored by the production of beta-galactosidase from a reporter construct containing the promoter region of the yeast STE3 gene and the coding region of the E. coli LacZ gene. Growth and progression through the cell cycle were also affected by Li+ and analysis of budded/unbudded ratios revealed that Li+ caused yeast cells to arrest in G1. These data provide support for the role of inositol phosphates in the mating response and cell cycle of Saccharomyces cerevisiae.

Cell Cycle↗

PubMed1995

The substrate-binding site in Cu nitrite reductase and its similarity to Zn carbonic anhydrase.

Here we investigate the structure of the two types of copper site in nitrite reductase from Alcaligenes xylosoxidans, the molecular organisation of the enzyme when the type-2 copper is absent, and its mode of substrate binding. X-ray absorption studies provide evidence for a fourth ligand at the type-2 Cu, that substrate binds to this site and indicates that this binding does not change the type-1 Cu centre. The substrate replaces a putative water ligand and is accommodated by a lengthening of the Cu-histidine bond by approximately 0.08 A. Modelling suggests a similarity between this unusual type-2 Cu site and the Zn site in carbonic anhydrase and that nitrite is anchored by hydrogen bonds to an unligated histidine present in the type-2 Cu cavity.

Absorptiometry, Photon↗