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Biomedical subjects

D A Johnson

Publications and source records attributed to D A Johnson.

At least 109 records · Page 6Linked to original sources
PubMed1996

Kinetic characterization of an organic radical in the ascarylose biosynthetic pathway.

The lipopolysaccharide of Yersinia pseudotuberculosis V includes a 3,6-dideoxyhexose, ascarylose, as the nonreducing end of the O-antigen tetrasaccharide. The C-3 deoxygenation of CDP-6-deoxy-L-threo-D-glycero-4-hexulose is a critical reaction in the biosynthesis of ascarylose. The first half of the reaction is a dehydration catalyzed by CDP-6-deoxy-L-threo-D-glycero-4-hexulose-3-dehydrase (E1), which is PMP-dependent and contains a redox-active [2Fe-2S] center. The second half is a reduction that requires an additional enzyme, CDP-6-deoxy-L-threo-D-glycero-4-hexulose-3-dehydrase reductase (E3, formerly known as CDP-6-deoxy-delta 3,4-glucoseen reductase), which has a FAD and a [2Fe-2S] center in the active site. Using NADH as the reductant in the coupled E1-E3 reaction, we have monitored the kinetics of a radical intermediate using both stopped-flow spectrophotometry and rapid freeze-quench EPR under aerobic and hypoxic conditions. In the EPR studies, a sharp signal at g = 2.003 was found to appear at a rate which is kinetically competent, reaching its maximum intensity at approximately 150 ms. Stopped-flow UV-vis analysis of the reaction elucidated a minimum of six optically distinguishable states in the mechanism of electron transfer from NADH to substrate. Interestingly, one of the detected intermediates has a time course nearly identical to that of the radical detected by rapid freeze-quench EPR. The difference UV-vis spectrum of this intermediate displays a maximum at 456 nm with a shoulder at 425 nm. Overall, these results are consistent with an electron transfer pathway that includes a radical intermediate with the unpaired spin localized on the substrate-cofactor complex. Evidence in support of this mechanism is presented in this report. These studies add the PMP-glucoseen radical to the growing list of mechanistically important bioorganic radical intermediates that have recently been discovered.

Bacterial Proteins↗
PubMed1996

Effects of dl-norephedrine and its enantiomers on norepinephrine uptake and release in isolated rat caudal artery.

Racemic norephedrine (dl-NOR), is a phenylethylamine derivative that is pharmacologically classified as a mixed-acting sympathomimetic amine. It is the active ingredient in many over-the-counter nasal decongestants and diet aid products. This research project was performed to examine the effects of dl-NOR and constituent enantiomers on the uptake and release of tritiated norepinephrine (3H-NE) in vascular tissue. All three NOR species significantly inhibited the uptake of 3H-NE into the isolated caudal artery of male Sprague-Dawley rats (p < 0.01). There was no significant difference in potency between either d- or l-NOR for this effect. There was, however, no effect of any NOR species on 3H-NE release, not even at concentrations which effectively inhibited uptake. These results indicate that inhibition of NE reuptake may be a component of the mechanism of action of NOR, but that a displacement of NE from neuronal storage sites is not. However, because the vasoactive effects of NOR have been shown to be mediated almost exclusively through the l-enantiomer, the lack of stereoselectivity in the current study indicates that the blockade of NE uptake by NOR is only a minor component of its vasoactive properties.

Adrenergic Uptake Inhibitors↗
PubMed1996

Inactivation of human lung tryptase: evidence for a re-activatable tetrameric intermediate and active monomers.

Human lung tryptase (HLT), a trypsin-like serine proteinase stored as an active enzyme in association with heparin in mast cell granules, is released into the extracellular environment when mast cells are activated. Tryptases are unusual in that they form tetramers and bind heparin. As there are no known endogenous tryptase inhibitors, loss of heparin and dissociation of the active tetrameric enzyme to inactive monomers has been proposed as the mechanism of control. Activity and intrinsic fluorescence were used to measure the stabilization of HLT by NaCl, glycerol, and heparin. At physiological salt concentrations in the absence of heparin, activity decayed rapidly (t1/2 = 1-4 min at 37 degrees C) to an intermediate that could be immediately reactivated by heparin. But protein structural changes, as measured by intrinsic fluorescence, were much slower (t1/2 = 16 min), indicating that the intermediate continued to exist as a tetramer that slowly changed to a monomer. HLT tetramers, either active or inactive, were stabilized by 2 M NaCl, 20% glycerol, and heparin. Maximum stabilization was obtained with approximately 1 mol of heparin per HLT subunit. Heparan sulfate also stabilized HLT activity and active HLT was bound to and recovered from cartilage. Subunits of the inactive intermediate appeared to be loosely associated as demonstrated by the rapid disappearance of the tetramer in gel filtration studies in 1 M NaCl (t1/2 = 1.8 min), but the tetramer was stable in lower ionic strength buffers containing heparin. Fluorescence anisotropy measurements in the absence of heparin were also consistent with a slow (t1/2 = 22 min) transition from tetramer to monomer, and native polyacrylamide gel electrophoresis provided additional evidence for a tetrameric intermediate. HLT monomers isolated by gel filtration were minimally active in the presence of heparin. These data show that heparin-free HLT rapidly converts to an "inactive", loose tetrameric intermediate that can be reactivated with heparin or slowly dissociate to less active monomers and that tryptase released from mast cells is likely to remain active in association with heparin or other extracellular components. Thus, tryptase affinity for glycosaminoglycans and substrate specificity limitations are the primary factors controlling the proteolytic functions of these enzymes.

Chromatography, Gel↗
PubMed1996

Medical Therapy of GERD: current state of the art.

A proton pump inhibitor is the most cost-effective choice for severe disease and, in low doses, for moderate disease. Patients with mild disease and infrequent relapses can be managed with a H2-receptor antagonist, an antacid, or cisapride.

Gastroesophageal Reflux↗
PubMed1996

Enhancement of hippocampal acetylcholine release by the neurosteroid dehydroepiandrosterone sulfate: an in vivo microdialysis study.

The effect of dehydroepiandrosterone sulfate (DHEAS) administered i.p. on the release of acetylcholine (ACh) from the hippocampus of anesthetized rats was examined using in vivo microdialysis. DHEAS significantly increased ACh release above the pre-treatment levels for all doses tested. The administration of 100 mg/kg significantly enhanced ACh release greater than 4-fold when compared to the saline-treated group 80 min following drug administration. This study is the first to demonstrate that the neurosteroid DHEAS, a negative allosteric modulator of the GABAA receptor, can enhance the release of ACh from neurons in the hippocampus.

Acetylcholine↗
PubMed1996

Kinetics of the reductive half-reaction of the iron-sulfur flavoenzyme CDP-6-deoxy-L-threo-D-glycero-4-hexulose-3-dehydrase reductase.

The conversion of CDP-4-keto-6-deoxy-D-glucose to CDP-4-keto-3,6-dideoxy-D-glucose is a key step in biosynthesis of ascarylose, the terminal dideoxyhexose of the O-antigen tetrasaccharide of the lipopolysaccharide from Yersinia pseudotuberculosis V. This transformation is catalyzed by two enzymes: CDP-6-deoxy-L-threo-D-glycero-4-hexulose-3-dehydrase (E1), which contains a pyridoxamine and a [2Fe-2S] center, and an NADH-dependent CDP-6-deoxy-L-threo-D-glycero-4-hexulose-3-dehydrase reductase (E3), which contains both an FAD and a [2Fe-2S] center. E1 reacts to form a Schiff base with CDP-4-keto-6-deoxy-D-glucose and catalyzes the elimination of the hydroxyl at position 3 of the glucose moiety, resulting in the formation of a covalently bound CDP-6-deoxy-delta(3,4)-glucoseen intermediate. E3 transfers electrons from NADH to E1, which uses these to reduce the delta(3,4)-glucoseen bond to produce CDP-4-keto-3,6-dideoxy-D-glucose. In this work, we have investigated the reductive half-reaction of E3 using both single wavelength and diode array stopped flow absorbance spectroscopy. We find that NADH binds to both oxidized (Kd = 52.5 +/- 2 microM) and two-electron-reduced (Kd = 12.1 +/- 1 microM) forms of E3. Hydride transfer from NADH to the FAD moiety occurs at 107.5 +/- 3 s-1 and exhibits a 10-fold deuterium isotope effect when (4R)-[2H]NADH is substituted for NADH. Following the hydride transfer reaction, NAD+ is released at 42.5 +/- 1 s-1 and electron transfer from the reduced FAD to the [2Fe-2S] center occurs rapidly. The extent of the intramolecular electron transfer reaction is pH-dependent with a pKa of 7.3 +/- 0.1, which may represent the ionization state of the N-1 position of the FAD hydroquinone of E3. Finally, E3 is converted to the three-electron-reduced state in a slow disproportionation reaction that consumes NADH: The [2Fe-2S] center of E3 was selectively disassembled by titration with mersalyl to give E3(apoFeS). The properties of this form of the enzyme are compared to those of the holoenzyme. Similarities and differences of the reductive half-reactions of E3 and related iron-sulfur flavoenzymes are discussed.

Cloning, Molecular↗
PubMed1996

Quinacrine noncompetitive inhibitor binding site localized on the Torpedo acetylcholine receptor in the open state.

Open-channel blockers of the nicotinic acetylcholine receptor (nAcChR) are widely thought to act sterically by entering and "plugging" the open channel of the nAcChR. However, quinacrine, a fluorescent open-channel blocker, has been recently shown to bind to the nAcChR at a site near the lipid bilayer while the receptor is in a closed, desensitized state, suggesting that at least one open-channel blocker might act allosterically outside the channel [Valenzuela et al. (1992) J. Biol. Chem. 267, 8238]. To determine whether or not quinacrine also binds near the lipid bilayer when the receptor is in an open state, a short-range lipophilic quencher (5-doxylstearate, 5-SA) was used to assess the proximity of the nAcChR-bound quinacrine to the lipid bilayer while the receptor was transiently open by an agonist. Initial experiments using a stopped-flow instrument established the conditions required to monitor a portion of the changes in quinacrine fluorescence associated with its binding to the receptor in the open state. 5-SA (80 microM) reduced the amplitude of the rapid agonist-induced change in quinacrine emission to 44% +/- 12% of the control value, indicating that the quinacrine was binding to a site proximal to the membrane-partitioned 5-SA. Control experiments established that 5-SA had no effect on the ability of the receptor to undergo agonist-induced conformational changes, suggesting that little, if any, 5-SA distributed into the channel lumen and perturbed the functional activity of the receptor. Together, the results indicate that quinacrine binds to a site on the open receptor that is in contact with the lipid bilayer and not in the channel lumen.

Animals↗
PubMed1996

Reproducibility of videokeratographic digital subtraction maps after excimer laser photorefractive keratectomy.

BACKGROUND: Digital subtraction photokeratography can best identify topographic changes after excimer laser photorefractive keratectomy (PRK). To evaluate the reproducibility of these topographic maps, the authors used a topographic modeling system to generate multiple subtraction maps from different combinations of technically acceptable preoperative and postoperative maps for eyes that underwent PRK. The assigned patterns for each patient then were evaluated for consistency. METHODS: Seven hundred twenty-two individual subtraction maps were generated for 64 eyes that underwent PRK. A mean of 11.3 maps were generated for each eye. The topography of each map was individually classified as normal, central island, peninsula, or asymmetric. All maps within a set (consisting of examinations for 1 patient at a single postoperative interval) then were examined as a unit to determine the overall topographic classification for that set of maps. Each set in which each constituent map had the same topographic assignment as the set was considered "nonvariant," whereas those sets in which one or more individual subtraction maps had different topographic assignments were considered "variant." RESULTS: Of the 64 sets, 33 (52%) were variant and 31 (48%) were nonvariant. CONCLUSIONS: Any one subtraction map produced by the topographic modeling system may not be a reliable indicator of the excimer effect.

Cornea↗
PubMed1996

Age and recovery from brain injury: clinical opinions and experimental evidence.

For many years the notion that brain damage causes less impairment in children than in adults (sometimes known as the 'Kennard Principle') has enjoyed widespread support among scientists and clinicians. More recently neuroscientists have questioned the Principle, most now taking an opposing view that damage to the rapidly developing brain can be more harmful than equivalent damage in adulthood. Many clinicians, however, appear reluctant to reject the Kennard Principle. This study investigates the extent to which the Kennard Principle still guides the judgement of different groups of health-care professionals (neurosurgeons, neurologists, neuropsychologists, general practitioners, nurses, physiotherapists, occupational therapists, and speech therapists). Subjects were asked to estimate the extent of recovery in clinically based but fictitious case studies which differed only in the reported age of the patient. The professions differed in their levels of optimism regarding the extent of recovery to be expected, but all predicted better recovery in younger patients (under 10) than in adults with otherwise similar brain injuries. The results are discussed in terms of their implications for the treatment of brain injuries in the young.

Adult↗
PubMed1996

Mitochondrial DNA restriction site map of Cochliomyia macellaria (Diptera:Calliphoridae).

The mitochondrial DNA of the secondary screwworm, Cochliomyia macellaria (F.), was cleaved with 15 restriction endonucleases to produce 54 restriction fragments. Forty-three restriction sites recognized by 12 enzymes were assigned positions on a restriction site map. Both the restriction fragments and the restriction site map were compared with published data from the screwworm, Cochliomyia hominivorax (Coquerel). The restriction site maps were aligned using a combination of highly conserved restriction sites. Estimated nucleotide divergence between the 2 species based on the fragments was 0.052. The divergence based on the restriction site map was 0.093. The data provide a baseline for future population or phylogenetic investigations.

Animals↗
PubMed1996

WAIS-R short-forms: criterion validity in healthy and clinical samples.

Regression equations developed by Crawford, Allan & Jack (1992) to estimate full-length WAIS-R IQs from seven short-forms were evaluated in healthy (N = 153) and neurological (N = 471) cross-validation samples. In the healthy sample the correlations between the short-forms and full-length IQs did not differ significantly from those obtained by Crawford et al. (1992). In the neurological sample the six- and seven-subtest short-forms, proposed by Crawford et al. (1992) and Warrington, James & Maciejewski (1986) respectively, differed from the other short-forms in that they had significantly higher criterion validity coefficients and did not systematically under- or overestimate IQs.

Adult↗
PubMed1996

The 5S rRNA gene units in ancestral two-rowed barley (Hordeum spontaneum C. Koch) and bulbous barley (H. bulbosum L.): sequence analysis and phylogenetic relationships with the 5S rDNA units of cultivated barley (H. vulgare L.).

5S rRNA genes from several accessions of Hordeum spontaneum and Hordeum bulbosum, wild relatives of cultivated barley, Hordeum vulgare, have been amplified by the polymerase chain reaction, cloned, and sequenced. Evaluation of aligned sequences along with principal coordinate analysis demonstrates that the two classes of 5S rDNA sequences found in cultivated barley, and subclasses (groups) of these sequences, can also be found in its closest wild relatives. The two classes of units, formerly categorized as containing short or long 5S rDNA repeats, are distinguishable by the presence or absence of a TAG repeating unit. Sequence comparisons of individual clones (units) isolated from different species have allowed us to confirm that orthology exists for several groups. This demonstration of orthologous groups suggests that the 5S rDNA sequence may be useful for further phylogenetic analysis in the genus Hordeum and possibly in the Triticeae.

Base Sequence↗
PubMed1996

Analysis of a hotspot for deletion formation within the intron of the chloroplast trnI gene.

The chloroplast genomes of higher plants encode several tRNA genes that contain highly conserved type II introns. Using primers specific to conserved 5' and 3' regions within the introns of the genes trnA (tRNA-ala) and trnI (tRNA-ile) we have PCR amplified parts of these introns from 36 plant species representing a wide range of plant families. Deletions were found in the introns of both tRNA genes. Fourteen species had detectable deletions within the intron of trnI and four species within the intron of trnA. The occurrence of these deletions among the various plant families suggests that the events leading to the formation of these deletions occurred independently many times during the evolution of higher plants. Analysis of the amplified PCR products from the trnI intron suggests that these independent deletions may not be random but appear to fall into two size classes. Several members of each class were cloned and sequenced and the end points of the deletions were mapped. The 3' ends of all deletions studied terminate within the same short region. The 5' ends of the deletions map to two different regions, giving rise to the two size classes. These two 5' deletion endpoint regions show some sequence similarity. Only two of the identified deletions contain directly repeated sequences at the deletion endpoints, a feature associated with homologous recombination. Our results suggest that within the trnI intron, there are preferred sites or "hotspots" for deletion formation involving a novel imprecise recombination mechanism. The significance of these sequences and possible mechanisms for deletion formation are discussed.

Base Sequence↗
PubMed1996

Hiatal hernia.

Hiatal hernia is a condition that is commonly encountered, widely discussed but frequently misunderstood. This review focuses on the pathogenesis, classification, associated conditions and complications of hiatal hernia. Additionally, the medical and surgical approaches to treatment are discussed with a detailed discussion of pre- and postoperative assessment in symptomatic patients.

Endoscopy, Gastrointestinal↗
PubMed1996

Ocular and orbital trauma from water balloon slingshots: a clinical, epidemiological, experimental, and theoretical study.

PURPOSE: To report the clinical findings of 17 patients with ocular/orbital injuries produced by launched water balloons; to determine water balloon kinetic energies in experimental and theoretical studies. METHODS: Six case histories are presented, 1 case was retrieved from the medical literature, and 10 cases were reported to the National Injury Information Clearinghouse of the United States Consumer Product Safety Commission. The energies were determined by field trials and calculations. RESULTS: Injuries included orbital contusions and hematomas, facial hypesthesia, eyelid lacerations, subconjunctival hemorrhages, corneal edema and abrasion, hyphemas, traumatic iritis, iris sphincter ruptures, iris atrophy, angle recession, iridodialyses, traumatic cataract, vitreous hemorrhages, retinal hemorrhages, macular hole formation, optic atrophy, and bony orbital wall fractures. Epidemiological analysis revealed that children and young adults, more often males, were injured, most commonly in the warm weather months (May through September). In field trials, maximum water balloon velocities ranged from 38 to 41 m/sec (85 to 92 mph) with kinetic energies from 176 to 245 J; by calculation, maximum velocities ranged from 42 to 54 m/sec (95 to 121 mph) with kinetic energies from 141 to 232 J. In a field demonstration a 300-g water balloon launched horizontally from a distance of 20 ft exploded a 12-kg watermelon. Classic physics calculations are presented to explain the complex bio-mechanical interactions between the water balloon and the eye. CONCLUSION: Kinetic energies of launched water balloons are comparable to or greater than kinetic energies experienced with a variety of common objects, including file bullets, which are well known to cause serious ocular and orbital injuries. In addition, these energies are far in excess of those required to perforate a cornea (0.7 to 1.7 J), rupture a globe (1 to 5.3 J), or fracture the bony orbit (1.8 to 14.7 j). Thus, this study demonstrates the serious and potentially vision- and life-threatening injuries inflicted by these "toys."

Adolescent↗
PubMed1996

Virtual reality: an assistive technology in neurological rehabilitation.

The need for improved neurological rehabilitation strategies is self-evident. Recent developments in the computer technology of virtual reality hold the promise of exciting progress in this area. In this paper four areas of potential application of virtual reality to neurological rehabilitation are reviewed.

Activities of Daily Living↗