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Biomedical subjects

D E Brown

Publications and source records attributed to D E Brown.

At least 19 recordsLinked to original sources
PubMed1992

Identification of topaquinone and its consensus sequence in copper amine oxidases.

The nature of the active site cofactor and the amino acid sequence flanking this structure have been determined in a range of copper amine oxidases. For enzymes from porcine plasma, porcine kidney, and pea seedlings, proteolytic digestion was performed on phenylhydrazone or p-nitrophenylhydrazone derivatives. Thermolysin treatment leads to relatively small active site peptides, which have been characterized by Edman degradation and by resonance Raman spectroscopy. Resonance Raman spectra of peptides show identical peak positions and intensities relative to each other and to a model p-nitrophenylhydrazone derivative of topaquinone hydantoin, establishing topaquinone as the cofactor in each instance. Edman degradation of peptides provides active site sequences for comparison to previous determinations with bovine serum and yeast amine oxidases. The available data establish a consensus sequence of Asn, Topa, Asp/Glu. Trypsin leads to significantly longer peptides, which reveal a high degree of sequence identity between plasma proteins from bovine and porcine sources (89%), with significantly decreased identity between the porcine serum and intracellular amine oxidases (56%). A lower degree of identity (45%) is observed between the pea seedling and mammalian enzymes. As an alternative to the isolation of active site peptides for topaquinone identification, visible spectra of intact proteins have been investigated. It is shown that p-nitrophenylhydrazone derivatives of native enzymes, active site-derived peptides, and a topaquinone model exhibit identical behavior, absorbing at 457-463 nm at neutral pH (pH 7.2) and at 575-587 nm in basic solution (1-2 M KOH). These spectral properties, which appear unique to topaquinone, provide a rapid and simple test for the presence of this cofactor in intact enzymes.(ABSTRACT TRUNCATED AT 250 WORDS)

Amine Oxidase (Copper-Containing)↗
PubMed1992

Evidence for copper and 3,4,6-trihydroxyphenylalanine quinone cofactors in an amine oxidase from the gram-negative bacterium Escherichia coli K-12.

The cofactors present in a amine oxidase induced in Escherichia coli K-12 by growth on 2-phenylethylamine have been studied by spectroscopic methods. E.s.r. spectroscopy establishes the presence of cupric copper while resonance Raman spectroscopy on the phenylhydrazine derivative of the enzyme provides strong evidence for the oxidized form of 3,4,6-trihydroxyphenylalanine (TOPA) quinone. The amine oxidase should accordingly be classified as EC 1.4.3.6. This is the first report of such an amine oxidase in a Gram-negative bacterium.

Amine Oxidase (Copper-Containing)↗
PubMed1992

The declining risk of post-transfusion hepatitis C virus infection.

BACKGROUND: The most common serious complication of blood transfusion is post-transfusion hepatitis from the hepatitis C virus (HCV). Blood banks now screen blood donors for surrogate markers of non-A, non-B hepatitis and antibodies to HCV, but the current risk of post-transfusion hepatitis C is unknown. METHODS: From 1985 through 1991, blood samples and medical information were obtained prospectively from patients before and at least six months after cardiac surgery. The stored serum samples were tested for antibodies to HCV by enzyme immunoassay, and by recombinant immunoblotting if positive. RESULTS: Of the 912 patients who received transfusions before donors were screened for surrogate markers, 35 seroconverted to HCV, for a risk of 3.84 percent per patient (0.45 percent per unit transfused). For the 976 patients who received transfusions after October 1986 with blood screened for surrogate markers, the risk of seroconversion was 1.54 percent per patient (0.19 percent per unit). For the 522 patients receiving transfusions since the addition in May 1990 of screening for antibodies to HCV, the risk was 0.57 percent per patient (0.03 percent per unit). The trend toward decreasing risk with increasingly stringent screening of donors was statistically significant (P less than 0.001). After we controlled for the method of donor screening, the risk of seroconversion was strongly associated (P less than 0.001) with the volume of blood transfused, but not with the use of particular blood components. CONCLUSIONS: The incidence of post-transfusion hepatitis C has decreased markedly since the implementation of donor screening for surrogate markers and antibodies to HCV. The current risk of post-transfusion hepatitis is about 3 per 10,000 units transfused.

Alanine Transaminase↗
PubMed1992

Tyrosine codon corresponds to topa quinone at the active site of copper amine oxidases.

The recently discovered organic cofactor of bovine serum amine oxidase, topa quinone, is an uncommon amino acid residue in the polypeptide backbone (Janes, S. M., Mu, D., Wemmer, D., Smith, A. J., Kaur, S., Maltby, D., Burlingame, A. L., and Klinman, J. P. (1990) Science 248, 981-987). The amine oxidase gene from the yeast Hansenula polymorpha has been cloned and sequenced (Bruinenberg, P. G., Evers, M., Waterham, H. R., Kuipers, J., Arnberg, A. C., and Geert, A. B. (1989) Biochim. Biophys. Acta 1008, 157-167). In order to understand the incorporation of topa quinone in eukaryotes, we have isolated yeast amine oxidase from H. polymorpha. Following protocols established with bovine serum amine oxidase, yeast amine oxidase was derivatized with [14C]phenylhydrazine, followed by thermolytic digestion and isolation of a dominant radiolabeled peptide by high pressure liquid chromatography. Comparison of resonance Raman spectra for this peptide to spectra of a model compound demonstrates that topa quinone is the cofactor. By alignment of a DNA-derived yeast amine oxidase sequence with the topa quinone-containing peptide sequence, it is found that the tyrosine codon, UAC, corresponds to topa quinone in the mature protein. In a similar manner, alignment of a tryptic peptide from bovine serum amine oxidase implicates tyrosine as the precursor to topa quinone in mammals.

Amine Oxidase (Copper-Containing)↗
PubMed1992

Occult fracture patterns of the knee associated with anterior cruciate ligament tears: assessment with MR imaging.

One hundred consecutive magnetic resonance (MR) images of the knee in patients with acute complete anterior cruciate ligament (ACL) tears were reviewed to evaluate the prevalence and patterns of associated occult fractures. Eighty-nine occult fractures were identified in 56 knees. All occult fractures were in the posterior aspect of the lateral tibial plateau. Of these, occult fractures were isolated in 24 cases (43%) and were in combination with fractures in the middle aspect of the lateral femoral condyle in 26 (46%), with fractures in the posterior aspect of the medial tibial plateau in four (7%), and with fractures involving all three areas in one (2%). Disruption of the ACL under valgus stress leads to anterior translation of the tibia and relative external rotation of the femur. This allows impaction of the posterior portion of the lateral tibial plateau against the middle of the lateral femoral condyle and accounts for the unique pattern of occult fractures associated with ACL tears. An occult fracture of the posterior lateral tibial plateau with or without an associated fracture in the lateral femoral condyle ("kissing contusion") is a relatively frequent finding in acute ACL tears and, when present, is highly suggestive of such an associated tear.

Adolescent↗
PubMed1992

Shoulder injuries.

Shoulder injuries result from acute or repetitive over-use mechanisms. An outline for a careful history and physical examination is presented along with recommendations for radiographic imaging of shoulder injuries dependent on the suspected diagnosis and injury mechanism. Descriptions of the more common shoulder problems are presented with recommendations for non-surgical treatment. Indications for surgical treatment that will allow the primary practitioner to best manage these injuries are presented.

Diagnosis, Differential↗
PubMed1991

The organic functional group in copper-containing amine oxidases. Resonance Raman spectra are consistent with the presence of topa quinone (6-hydroxydopa quinone) in the active site.

Resonance Raman spectroscopy has been used to probe the structure of the organic cofactor in copper-containing amine oxidases from bovine plasma, porcine kidney, pea seedlings, and the bacterium Arthrobacter P1. The enzymes were first derivatized with phenylhydrazine or p-nitrophenylhydrazine; resonance Raman spectra were obtained on the intact derivatized enzymes and on a derivatized active-site peptide isolated from bovine plasma amine oxidase. Spectra of the intact amine oxidase phenylhydrazones are practically identical, consistent with the enzymes examined containing a similar cofactor. Only minor frequency shifts and some intensity variations are detected between the resonance Raman spectra of intact bovine plasma amine oxidase and the isolated peptide. These spectral perturbations are attributable to differences in the micro-environment between the intact, folded protein and the isolated small peptide in aqueous solution. This rules out the possibility that a new structure is formed during the isolation of the derivatized active-site peptide. Importantly, the resonance Raman spectra of the phenylhydrazine and p-nitrophenylhydrazine derivatives of the bovine plasma amine oxidase peptide are identical to the spectra of the corresponding derivatives of topa quinone (6-hydroxydopa quinone). Hence these data provide strong, independent support for the recent identification of topa as the organic functional group in bovine plasma amine oxidase (Janes, S. M., Mu, D., Wemmer, D., Smith, A. J., Kaur, S., Maltby, D., Burlingame, A. L., and Klinman, J.P. (1990) Science 248, 981-987).

Amine Oxidase (Copper-Containing)↗
PubMed1991

A Cu(I)-semiquinone state in substrate-reduced amine oxidases.

The role of copper in copper-containing amine oxidases has long been a source of debate and uncertainty. Numerous electron paramagnetic resonance (EPR) experiments, including rapid freeze-quench studies, have failed to detect changes in the copper oxidation state in the presence of substrate amines. One suggestion that copper reduction might occur, has never been confirmed. Copper amine oxidases contain another cofactor, recently identified as 6-hydroxydopa quinone (topa quinone), which is reduced by substrates. Copper has been implicated in the reoxidation of the substrate-reduced enzyme, but the failure to detect any copper redox change has led to proposals that Cu(II) acts as a Lewis acid, that it has an indirect role in catalysis, or that it serves a structural role. We present evidence for the generation of a Cu(I)-semiquinone state by substrate reduction of several amine oxidases under anaerobic conditions, and suggest that the Cu(I)-semiquinone may be the catalytic intermediate that reacts directly with oxygen.

Amine Oxidase (Copper-Containing)↗
PubMed1991

Resonance Raman spectroscopy of methylamine dehydrogenase from bacterium W3A1.

Resonance Raman spectroscopy has been used to probe the structure of the covalently bound quinone cofactor in methylamine dehydrogenase from the bacterium W3A1. Spectra were obtained on the phenylhydrazine and 2-pyridylhydrazine derivatives of the native enzyme, on the quinone-containing subunit labeled with phenylhydrazine, and on an active-site peptide also labeled with phenylhydrazine. Comparisons of these spectra to the corresponding spectra of copper-containing amine oxidase derivatives indicate that the quinones in these two classes of quinoproteins are not identical. The resonance Raman spectra of the native enzyme and small subunit have also been measured. 16O/18O exchange permitted the carbonyl modes of the quinone to be identified in the resonance Raman spectrum of oxidized methylamine dehydrogenase: a band at 1614 cm-1, together with a shoulder at 1630 cm-1, are assigned as modes containing substantial C = O stretching character. D2O/H2O exchange has pronounced effects on the resonance Raman spectrum of the oxidized enzyme, suggesting that the quinone may have numerous hydrogen bonds to the protein or that it is unusually sensitive to the local environment. Resonance Raman spectra of the isolated small subunit, and its phenylhydrazine derivative, are considerably different from the corresponding spectra of the intact protein. An attractive explanation for these observations is that the quinone cofactor in methylamine dehydrogenase from W3A1 is located at the interface between the large and small subunits, as found for the enzyme from Thiobacillus versutus [Vellieux, F. M. D., Huitema, F., Groendijk, H., Kalk, K. H., Frank, J. Jzn., Jongejan, J. A., & Duine, J. A. (1989) EMBO J. 8, 2171-2178].(ABSTRACT TRUNCATED AT 250 WORDS)

Bacteria↗
PubMed1991

An optically scanned EMS reporting form and analysis system for statewide use: development and five years' experience.

Analysis of emergency medical services (EMS) systems data is crucial to planning, education, research, and quality assurance programs. Currently, comparative analysis of EMS data between regions or states is virtually impossible due to wide variations in data collection and analysis methods. To devise a practical and uniform EMS reporting system, we referenced the minimum data set (MDS) established by the federal government in 1974 and surveyed 22 states known to be using uniform reporting systems. In developing our final data set, elements were added based on inclusion in the MDS, national survey results, a review of current EMS literature, and consensus of local EMS providers. This set of 48 elements then was incorporated into a reporting form using narrative and optically scanned formats, allowing automated data collection for computer analysis. After a pilot study, the system was improved to allow high-speed ink reading and large volume data storage and analysis using a microcomputer. This system has subsequently been adopted by seven states. The combined data base exceeds 250,000 cases. Error screening algorithms ensure data integrity and are also used for quality assurance. Customized output reports can be generated within minutes and have assisted in EMS quality assurance, planning, and research. We believe that the successful performance of this system supports the use of the suggested data elements as well as optical scanning and microcomputer analysis of EMS data.

Data Collection↗
PubMed1991

MR of the knee: the significance of high signal in the meniscus that does not clearly extend to the surface.

On MR images of the knee it is sometimes impossible to determine with confidence if a focus of high signal in the meniscus is confined to the substance of the meniscus or if it extends to involve the surface. This is a critical differentiation because the latter represents meniscal tears that can be found and treated at arthroscopy, whereas the former represents degeneration, tears, or perhaps normal variants that cannot be detected or treated arthroscopically. We make an equivocal diagnosis of a tear when it is difficult to decide if signal in a meniscus involves the meniscal surface. We studied MR scans of the knee in 142 consecutive patients for the presence of such equivocal tears. Their prevalence was 14% (20/142); 17 were in the posterior horn of the lateral meniscus and three were in the posterior horn of the medial meniscus. In 13 cases with arthroscopy/arthrotomy correlation, no tears were found. In one of the 20 patients in whom the meniscus was removed during arthroplasty, histologic examination of the meniscus showed separation of collagen bundles, which was caused by meniscal degeneration confined to the substance of the meniscus. These results suggest that a meniscal tear is unlikely when MR scans show a focus of high signal in a meniscus that does not unequivocally extend to involve the surface of the meniscus.

Adolescent↗
PubMed1991

Maternal endocrine and fetal metabolic responses to heat stress.

Heat-stressed pregnant ewes deliver intrauterine growth-retarded lambs. Selected maternal and fetal changes were investigated during acute heat stress in order to elucidate the mechanism for this growth retardation. Uterine blood flow decreased 20 to 30% in pregnant ewes during 1 degree C increases in core temperature. The decreases were accompanied by 60 and 100% increases in serum oxytocin and antidiuretic hormone, respectively. These effects were mimicked by salt loading or injections of antidiuretic hormone or oxytocin, suggesting a role for either or both hormones in regulating uterine blood flow during pregnancy. Chronically heat-stressed pregnant ewes were delivered by Caesarean section. Their fetuses were approximately 20% smaller than thermoneutral controls. Within each pair of heat-stressed twins, one fetus weighted one-third less than its litter mate. No difference in weights were observed within the control twins. The livers and brains of the heat-stressed fetuses were disproportionate in size. The livers from the small heat-stressed twins contained only one-half the protein of the controls and one-fourth the protein of their litter mates. Muscle protein was decreased in the heat-stressed fetuses, and liver and muscle glycogen were elevated as were liver arginase, glutamate-pyruvate transaminase and muscle creatinine. These results are consistent with the following hypothesis: heat stress stimulates the release of maternal antidiuretic hormone or oxytocin, which reduces uterine blood flow and causes a shift in fetal metabolism from anabolic to catabolic pathways; one fetus of heat-stressed twins is more severely affected than its litter mate.

Animals↗
PubMed1990

Single-exposure dual-energy computed radiography: improved detection and processing.

Recent reports have emphasized the potential for dual-energy computed radiographic applications. An improved method for single-exposure material-selective imaging with a photostimulable phosphor computed radiography system was investigated. The essential elements of the technique are (a) prefiltration with gadolinium, which divides the incident broad-beam x-ray spectrum into low-energy and high-energy peaks; (b) a cassette consisting of four photostimulable phosphor plates that record images of increasing mean energies, with a computed energy separation of 23 keV from the front to the rear plate; (c) spatially dependent scatter and beam-hardening corrections; and (d) a noise-reduction algorithm based on noise correlations between bone-selective and soft-tissue-selective dual-energy images. These elements result in improved material cancellation and signal-to-noise ratio throughout the image.

Algorithms↗
PubMed1988

Preparation of serum oxytocin and arginine vasopressin prior to radioimmunoassay: simultaneous extraction and separation on C18 Sep-Pak cartridges.

Oxytocin (OT) and arginine vasopressin (AVP) are often secreted in response to the same stimuli. The hormones are seldom assayed together, however, because of labor intensive sample preparation and the duplicate volumes required. A method has been developed for the simultaneous extraction and separation of OT and AVP from a single serum sample. The method is suited for sample preparation prior to radioimmunoassay (RIA) and reduces sample volume and processing time by 50%. Serum, supplemented with labeled and unlabeled OT and AVP, was adsorbed onto C18 (octadecasilyl-silica, ODS) Sep-Pak cartridges. After washing with phosphosaline and 3% aqueous acetone, OT was eluted with 98% aqueous acetone followed by AVP with 80% acidified (0.02 mol/L HCl) acetone. The recoveries, determined by radioactivity and RIA measurements, were 86 +/- 3% (OT) and 71 +/- 7% (AVP). Cross contamination was less than 10%. Sep-Paks extracted up to 100 pg/mL of the hormones from 10 mL of serum. The method was employed to measure OT and AVP in the pregnant ewe. Both hormones were elevated during salt-loading and dehydration and were decreased by carotid infusions of ethanol.

Animals↗
PubMed1988

Effect of blood sampling and shipment to slaughter on plasma catecholamine concentrations in bulls.

The effect of venipuncture on blood serum catecholamine concentrations was studied in five yearling Red Angus bulls. Treatments consisted of sampling jugular vein blood via indwelling cannulas in a squeeze chute every 10 min for 1 h; sampling jugular vein blood from an indwelling catheter before, during and after venipuncture simulation; sampling jugular vein blood by venipuncture in a chute after shipment of 320 km to a slaughter plant; and sampling pooled blood from the neck during exsanguination at slaughter. Plasma epinephrine (E) and norepinephrine (NE) concentrations were determined by high performance liquid chromatography (HPLC) with electrochemical detection (ECD). Prevenipuncture NE and E in blood sampled from an indwelling catheter extended through a solid wall were 1.0 and .5 pmol/ml plasma, respectively. Plasma NE and E during venipuncture (1.6 and 1.1 pmol/ml, respectively) and immediately after cannulation in a squeeze chute (1.8 and 1.2 pmol/ml, respectively) were higher than prevenipuncture concentrations. Plasma NE and E, 1.9 and 1.2 pmol/ml plasma after shipment of 320 km, increased during exsanguination to 135.8 and 81.3 pmol/ml plasma, respectively. The NE and E in blood samples collected by venipuncture in a chute or box stall do not represent resting concentrations of these catecholamines.

Animals↗
PubMed1988

Reversal of depressed miduterine arterial flow during hyperthermia-induced respiratory alkalosis.

The influence of ambient and arterial PCO2 on miduterine arterial flow of pregnant sheep acutely exposed to hot environments was investigated. Five mixed-breed ewes between 120 and 130 days of gestation were subjected to hot environments (increasing from thermoneutral 23 to 40 degrees C), and arterial blood pH, PCO2, and PO2 were determined at 5-min intervals. Respiratory rate, heart rate, rectal temperature, blood pressure, and miduterine arterial flow were continuously monitored prior to and during elevation of ambient air temperature. When miduterine arterial flow had decreased to 50% of thermoneutral control levels, ambient air CO2 was increased to 2.5%. Elevated ambient inspired CO2 caused a reversal in arterial pH and PCO2 to near thermoneutral levels. Miduterine arterial flow increased to 77% of the control levels following the elevated ambient PCO2 period. Respiratory rate also decreased when ambient CO2 was increased but remained 136% greater than the thermoneutral control level. All other parameters remained near their heat stress (40 degrees C) level during the elevation of ambient CO2. These data indicate that heat-stress-induced depression of miduterine arterial flow is vasoactively regulated, and cause-effect related to both arterial pH and PCO2, and thermoregulatory shunting of blood to heat-dissipating surfaces.

Alkalosis, Respiratory↗
PubMed1987

Bupivacaine disposition in mother, fetus, and neonate after spinal anesthesia for cesarean section.

Uptake of bupivacaine from the subarachnoid space and its placental transfer were measured in six patients undergoing elective cesarean section. Maternal plasma levels (59 +/- 32 ng/ml) were only about 5% of those found in a comparable previous study of epidural anesthesia. Mean plasma umbilical venous bupivacaine levels (20.2 +/- 21 ng/ml) were 7% of those found after epidural anesthesia. Mean umbilical venous/maternal venous bupivacaine ratios were 0.34 +/- 0.12 and mean umbilical arterial/umbilical venous ratios were 0.81 +/- 0.30. No bupivacaine was detectable in neonatal plasma 24 hr after delivery. Neonatal urine had measurable levels of both bupivacaine and its inactive metabolite, 2,6-pipecolylxylidine (PPX), for at least 36 hr after delivery. The results demonstrate that bupivacaine crosses the placenta and reaches the fetus, but in very low amounts. This transplacental passage occurs despite injection of only small doses of a very highly protein bound drug into the subarachnoid space.

Anesthesia, Obstetrical↗