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Biomedical subjects

M E Johnson

Publications and source records attributed to M E Johnson.

At least 109 records · Page 6Linked to original sources
PubMed1994

Comparison of protein structures in solution using local conformations derived from NMR data: application to cytochrome c.

Structural comparisons of proteins in solution are often required to examine structure-functional relationships, study structural effects of mutations or distinguish between various forms of the same molecule under different conditions. A nuclear magnetic resonance (NMR) based probabilistic strategy is presented and used to study the structural differences between the two redox states of cytochrome c in solution. A probabilistic approach is employed to calculate the main chain conformations of horse ferro- and ferricytochrome c in solution, based on the published sequential d connectivity data. Conformational differences between the two oxidation states of horse cytochrome c in solution are found to be statistically significant. The largest changes in conformation are at residues Lys27, Thr28, Leu32, Gln42, Thr47, Tyr48, Thr49, Glu69, Lys72, Met80, Phe82, Ile85 and Lys86, all of which are close to the heme (within 14 A of the heme iron in the high resolution Xray structure of tuna cytochrome c). We suggest that these conformational changes may modulate local dipole moments and hence influence the interactions of cytochrome c with its physiological redox partners during the electron transfer process. The oxidation state dependent conformational differences are found to be much greater in solution than in the crystalline state, and the solution and crystal structures differ significantly in regions close to the heme. These results suggest that the highly charged nature of cytochrome c makes this protein particularly sensitive to the ionic strength of its environment and leads to differences between crystal and solution structures in the same oxidation state. In such cases, crystal structures must be used with caution for modeling molecular interactions in vivo. More generally, this analysis indicates that the determination of accurate local conformations based on nmr data can provide useful information about structure-functional aspects of proteins in solution.

Amino Acid Sequence↗
PubMed1994

Dapiprazole clinical efficiency for counteracting tropicamide 1%.

We evaluated the clinical usefulness of dapiprazole in reversing the effects of tropicamide 1.0%. Our study was random, masked with placebo, and used one eye of each subject as a control. Thirty subjects were given dapiprazole as directed by the manufacturer 30 min after being dilated by one drop each of proparacaine 0.5%, tropicamide 1.0%, and then 5 min later another drop of tropicamide 1.0%. Pupil diameter, amplitude of accommodation, conjunctival injection, and intraocular pressure were evaluated. Each of these variables was measured: (1) before instillation of the diagnostic agents; (2) before the instillation of dapiprazole; and (3) at 30, 60, 120, and 180 min after the final instillation of dapiprazole. The average pupillary recovery time for dapiprazole-treated eyes was significantly less than for nontreated eyes. Accommodation also showed faster recovery. Comfortable reading ability returned after approximately 43 min with dapiprazole vs. 66 min without dapiprazole. All of our subjects exhibited conjunctival hyperemia after the administration of dapiprazole. This persisted throughout the 180 min observation period after its administration.

Accommodation, Ocular↗
PubMed1994

A psychiatric hospital 100 years ago: II. Patients, treatment, and daily life.

Data from archival sources were used to determine the kinds of patients treated at the St. Louis City (later St. Louis County) Insane Asylum, the treatments they received, activities of daily life in the asylum, and political factors affecting operation of the asylum in the late 19th and early 20th centuries. Data from patient records, journal and newspaper articles, and annual reports of asylum superintendents from the period were analyzed. The authors conclude that although much has changed in the operation of public psychiatric hospitals in the past 100 years, some themes, including inappropriate referrals of forensic cases to psychiatric hospitals, problems of discharging long-stay patients, and the media's tendency to sensationalize events and conditions in hospitals, remain the same.

Activities of Daily Living↗
PubMed1994

Risk factors in patients having surgery to create an arteriovenous fistula.

Patients requiring creation of an arteriovenous (AV) fistula for long-term dialysis access have a high incidence of cardiovascular risk factors, as well as immunosuppression, neuropathy, and platelet dysfunction related to renal failure. However, the morbidity and mortality of AV fistula creation, and the effect of anesthetic technique on outcome, have not been reported. We therefore retrospectively studied all patients (n = 469) having an AV fistula placed at the Mayo Clinic between 1986 and 1991. Preoperative variables studied were age (mean 63 +/- 14 yr), sex (60% male), diabetes mellitus (26% incidence), hypertension (92%), the presence of a previous AV fistula (31%), coronary artery disease (86%), and previous myocardial infarction (MI) (42%). Outcome variables studied were fistula failure prior to first attempted dialysis (8.7% incidence), infection (3.4%), neuropathy (1.9%), seizure (0.2%), nonfatal cardiac event (MI or arrest) (1.5%), and fatal cardiac event (2.1%). Associations of preoperative factors, outcomes, and anesthetic technique were analyzed by analysis of variance and Wilcoxon rank sum analysis for age, and by exact conditional frequency table analysis for all other factors. A previous AV fistula was associated with infection (P < 0.002) and nonfatal cardiac events (P < 0.003). Increased age (P < 0.025) and previous MI (P < 0.01) were associated with adverse cardiac outcomes. Neither local anesthesia, brachial plexus block, nor general anesthesia were significantly associated with an increased frequency of any adverse outcome. The comparison of general with local and brachial plexus anesthetics was limited by low statistical power.(ABSTRACT TRUNCATED AT 250 WORDS)

Aged↗
PubMed1994

The cost effectiveness of Air Force optometry.

A study was performed which attempted to assess the relative value of a military optometric practice and to provide epidemiological information on ocular pathologies typical in that practice setting. A mock billing system was employed which assessed fees against patient examinations and procedures using Current Procedural Terminology codes. The fees were determined from a survey of the fee structures of local optometrists. The study showed that the military optometric practice was cost effective in relation to the cost that would be incurred through civilian referral. It also demonstrated that military optometric practice is full in scope and encounters a wide variety of pathological entities above and beyond standard refractive modalities.

Aerospace Medicine↗
PubMed1994

Cytosolic free calcium and cell death during metabolic inhibition in a neuronal cell line.

Elevated free cytosolic Ca2+ (Ca2+i) has been implicated as a mechanism of hypoxic neuronal death. The calcium hypothesis postulates that the basic metabolic response to hypoxic ATP depletion is a toxic increase in free cytosolic Ca2+i in all cell types. This inherent response then creates the environment in which subsequent derangements of Ca2+i may occur, for example, from glutamate excitotoxicity. Although the effect of glutamate on neuronal Ca2+i has been extensively studied, the basic neuronal response to hypoxia independent of glutamate receptor activation is not well defined. We therefore assayed both Ca2+i and plasma membrane integrity in fura-2-loaded, single SK-N-SH neuroblastoma cells, using digitized video microscopy and metabolic inhibition (2.5 mM NaCN, 10 mM 2-deoxyglucose) to model the ATP depletion of hypoxia. Median time to cell death was 90 min (n = 51 cells). Initial Ca2+i was 121 +/- 67 nM. Ca2+i increased by 50 nM after 5-10 min of metabolic inhibition. Blebbing of the cell membrane was evident within 30 min. Ca2+i did not appreciably increase further until the time of cell death, when the loss of plasma membrane integrity allowed unimpeded influx of extracellular Ca2+.(ABSTRACT TRUNCATED AT 250 WORDS)

Calcium↗
PubMed1993

1H NMR-based determination of the three-dimensional structure of the human plasma fibronectin fragment containing inter-chain disulfide bonds.

Human plasma fibronectin is a plasma glycoprotein that plays an important role in many biological processes. It consists of two identical 230-250-kDa subunits that are joined by two disulfide bonds near their carboxyl termini. Each subunit contains various binding domains composed of three types of homologous repeats. Recent work has determined the three-dimensional structures of various repeat fragments, but little is known about the three-dimensional structure of the carboxyl-terminal region. A recent NMR study of a plasmin-digested carboxyl-terminal inter-chain disulfide-linked heptapeptide dimer has proposed that the two subunits are arranged in an antiparallel fashion (An et al. (1992) Biochemistry 31, 9927-9933). We have now determined the three-dimensional structure for a substantial portion of a trypsin-digested interchain disulfide-linked 52-residue (6 kDa) fragment of the carboxyl-terminal of human plasma fibronectin (which includes the above-mentioned heptapeptide dimer) using two-dimensional NMR methods and a new strategy for NMR-based protein structure determination. The NMR data requires that the two chains in the dimer be linked in a symmetric, antiparallel arrangement. The resulting monomer conformation consists of two twisted or coiled segments, Thr3-Asn6 and Ile9-Phe12, connected by the Cys7-Pro8 residues in extended conformations, with the two monomer chains cross-linked at residues Cys7 and Cys11. The conformation of the heptapeptide dimer region differs substantially from the conformation proposed by An et al.

Amino Acid Sequence↗
PubMed1993

Rapid sizing of individual fluorescently stained DNA fragments by flow cytometry.

Large, fluorescently stained restriction fragments of lambda phage DNA are sized by passing individual fragments through a focused continuous wave laser beam in an ultrasensitive flow cytometer at a rate of 60 fragments per second. The size of the fluorescence burst emitted by each stained DNA fragment, as it passes through the laser beam, is measured in one millisecond. One hundred sixty four seconds of fluorescence burst data allow linear sizing of DNA with an accuracy of better than two percent over a range of 10 to 50 kbp. This corresponds to analyzing less than 1 pg of DNA. Sizing of DNA fragments by this approach is much faster, requires much less DNA, and can potentially analyze large fragments with better resolution and accuracy than with gel-based electrophoresis.

Bacteriophage lambda↗
PubMed1993

Molecular dynamics of sickle and normal hemoglobins.

Molecular dynamics (MD) simulations have been carried out for 62.5 ps on crystal structures of deoxy sickle cell hemoglobin (HbS) and normal deoxy hemoglobin (HbA) using the CHARMM MD algorithm, with a time step of 0.001 ps. In the trajectory analysis of the 12.5-62.5 (50 ps) simulation, oscillations of the radius of gyration and solvent-accessible surface area were calculated. HbS exhibited a general contraction during the simulation, while HbA exhibited a nearly constant size. The average deviations of simulated structures from the starting structures were found to be 1.8 A for HbA and 2.3 A for HbS. The average rms amplitudes of atomic motions (atomic flexibility) were about 0.7 A HbA and about 1.0 A for HbS. The amplitudes of backbone motion correlate well with temperature factors derived from x-ray crystallography. A comparison of flexibility between the alpha- and beta-chains in both HbA and HbS indicates that the beta-chains generally exhibited greater flexibility than the alpha-chains, and that the HbS beta-chains exhibit greater flexibility in the N-terminal and D- and F-helix regions than do those of HbA. The average amplitude of backbone torsional oscillations was about 9 degrees, a value comparable with that of other simulations, with enhanced torsional oscillation occurring primarily at the ends of helices or in loop regions between helices. Comparison of atomic flexibility and torsional oscillation results suggests that the increased beta-chain flexibility results from relatively concerted motions of secondary structure elements. The increased flexibility may play an important role in HbS polymerization.(ABSTRACT TRUNCATED AT 250 WORDS)

Computer Simulation↗
PubMed1993

Cytotoxic and antimalarial bisbenzylisoquinoline alkaloids from Cyclea barbata.

An alkaloid extract derived from the roots of Cyclea barbata demonstrated cytotoxic and antimalarial activities, and five bisbenzylisoquinoline alkaloids, (+)-tetrandrine [1], (-)-limacine [2], (+)-thalrugosine [3], (+)-homoaromoline [4], and (-)-cycleapeltine [5], were isolated as the active principles. The complete and unambiguous assignments of the 1H- and 13C-nmr data of these substances were made by 1D and 2D nmr techniques (COSY, phase-sensitive ROESY, HETCOR, and FLOCK).

Alkaloids↗
PubMed1993

Sex role orientation and expectations about counseling.

We examined the relationship between sex role orientation and expectations about counseling in 121 female and 127 male college students who completed the Bem (1974) Sex Role Inventory and the Expectations About Counseling Questionnaire (Tinsley, Workman, & Kass, 1980). Using multiple regression analyses, we identified significant predictors for 15 of the 17 scales, with femininity accounting for most of the explained variance. Gender accounted for little variance across all variables.

Adolescent↗
PubMed1993

Effect of halothane on hypoxic toxicity and glutathione status in cultured rat hepatocytes.

BACKGROUND: In hypoxic rats, halothane causes hepatotoxicity at oxygen levels that would cause minimal hepatotoxicity in the absence of halothane. Using a model that excludes systemic and extrahepatic effects of halothane, the authors tested the hypothesis that halothane hepatotoxicity in the whole-rat model is caused by a direct hepatotoxic effect of halothane, which is mediated by halothane-derived free radicals. METHODS: Rat hepatocyte monolayer cultures were exposed to defined gas phases for 2 h. Three experimental variables were present or absent: hypoxia (1% O2), halothane (2%), and cytochrome P-450 induction (by phenobarbital). Two experimental outcomes were measured: aspartate aminotransferase release, a measure of cell death, and reduced glutathione, an endogenous free radical scavenger whose levels are decreased by physiologically significant free radical injury. RESULTS: As anticipated, hypoxia increased cell death. Cytochrome P-450 induction by itself increased cell death during hypoxia. However, halothane had no effect on cell death during hypoxia, with or without cytochrome P-450 induction. Halothane had no toxic effect, even when glutathione was depleted before the onset of hypoxia. Glutathione was decreased moderately by hypoxia alone. Neither halothane nor cytochrome P-450 induction had any effect on glutathione levels. CONCLUSIONS: Halothane was not toxic, and it did not generate a physiologically significant free radical insult during hypoxia in the isolated rat hepatocyte under the experimental conditions used in testing.

Animals↗
PubMed1993

Efficacy of dapiprazole.

A study was performed to evaluate the clinical usefulness of dapiprazole in a private clinical setting. This study was unmasked, but used one eye of each subject as a control. Thirty consecutive subjects were given dapiprazole as directed by the manufacturer. The administration of dapiprazole followed bilateral dilation for routine fundus examination. Subjects were dilated using one drop each of proparacaine 0.5%, tropicamide 1.0%, and phenylephrine 2.5%. Pupil diameter, amplitude of accommodation, and conjunctival injection were evaluated. Each of these three variables was measured (1) before instillation of the diagnostic agents, (2) before the instillation of diapiprazole, and (3) at 30, 60, 120, and 180 min after the final instillation of dapiprazole. The average pupillary recovery time for dapiprazole-treated eyes was similar to previously published data. Accommodation also showed significant recovery, with comfortable reading ability returning after approximately 30 min. The design of our study did not permit us to determine what portion of accommodation recovery was attributable to alpha ciliary muscle effect and what portion resulted from the increased depth of field that was due to the pupillary constriction. All of our subjects exhibited conjunctival hyperemia after the administration of dapiprazole. This side effect persisted through the entire 180-min observation period that followed dapiprazole administration.

Accommodation, Ocular↗
PubMed1993

Efficacy of dapiprazole with hydroxyamphetamine hydrobromide and tropicamide.

BACKGROUND: Clinical interest in ophthalmic agents to reverse pharmacologically induced mydriasis safely has been attempted for over fifty years. These agents may aid practitioners in assisting the younger patient to regain unable near visual function. METHODS: We evaluated the usefulness of dapiprazole in a private clinical setting. Our study was doubled masked, randomized and used one eye of each subject as a control. Twenty-six consecutive subjects were given dapiprazole as directed by its manufacturer. This administration of dapiprazole followed bilateral pupil dilation with proparacaine 0.5%, hydroxyamphetamine 1.0% and tropicamide 0.25%. Pupil diameter, amplitude of accommodation, intraocular pressure and conjunctival injection were each evaluated prior to instillation of the diagnostic agents, immediately before the instillation of dapiprazole and at 30, 60, 120, and 180 minutes after the final instillation of dapiprazole. RESULTS: The average pupillary recovery time for dapiprazole-treated eyes was similar to previously published data. Accommodation also showed significant recovery, with comfortable reading ability returning by approximately 45 minutes. CONCLUSION: The design of our study did not permit us to determine what portion of accommodation recovery if any was attributable to a ciliary muscle effect and what portion may have resulted from the increased depth of field attending pupillary constriction.

Accommodation, Ocular↗
PubMed1992

Bivalent-metal binding to CheY protein. Effect on protein conformation.

CheY is a 14 kDa cytoplasmic protein that is activated by the transfer of a phosphoryl moiety to Asp-57 from phosphoCheA during signal transduction in bacterial chemotaxis. It has been established that metal ions are necessary for the autophosphorylation of CheA, the transfer of phosphate from phosphoCheA to CheY and the autodephosphorylation of phosphoCheY. In this work, paramagnetic relaxation enhancement has been used in conjunction with one- and two-dimensional n.m.r. to study the interaction of CheY with bivalent metal ions. These studies have led to the discovery of two conformations of the protein in water, corresponding to the metal-free and the metal-bound states. Binding of bivalent cations like Mg2+, Ca2+, Sr2+, Zn2+ and Mn2+ results in a conformational change from the metal-free to the metal-bound state. Preliminary assignments of the aromatic proton resonances are reported. Comparison of phase-sensitive double-quantum-filtered COSY, homonuclear Hartmann-Hahn coherence transfer and nuclear Overhauser enhancement spectra from the metal-bound and metal-free protein indicates that Trp-58, Thr-87 and Tyr-106 are particularly affected by the conformational change involved, and that this change is limited to a small number of residues. In addition, homonuclear Hartmann-Hahn coherence transfer experiments with paramagnetic Mn2+ show significant suppression of cross-peaks associated with Trp-58 and several neighbouring residues. Comparison of the distances estimated using n.m.r. with the CheY crystal structure indicates that the n.m.r. results are consistent with bivalent metal binding at the cluster of aspartic acid residues that includes Asp-13 and Asp-57. These studies also demonstrate the utility of paramagnetic metal-induced relaxation in conjunction with two-dimensional n.m.r. measurements for exploring ligand-binding sites.

Amino Acid Sequence↗
PubMed1992

Specificity and affinity of binding of phosphate-containing compounds to CheY protein.

1H- and 31P-n.m.r. have been used to study the interaction of the bacterial chemotaxis protein, CheY, with ATP and a variety of other phosphates in the presence and absence of bivalent metal ions. In the metal-bound conformation, CheY will bind nucleotide phosphates and phosphates in general, while in the metal-free conformation CheY loses its affinity for phosphates. In the presence of low concentrations of nitroxide-spin-labelled ATP (SL-ATP), specific proton resonances of metal-bound CheY are suppressed, indicating that ATP binds to a specific site on this metal-bound form of the protein. These studies also show that the same resonances are affected by the binding of SL-ATP and Mn2+, indicating that the phosphate- and metal-binding sites are close to each other and to Asp-57 (the site of phosphorylation in CheY). 1H- and 31P-n.m.r. studies using ATP, GTP, TTP, UTP, ADP, AMP and inorganic phosphates show that the binding is not specific for adenine, and does not involve the base directly, but is mediated primarily by the phosphate groups. Experiments with a phosphorylation mutant (Asp-13-->Asn) suggest that the observed phosphate binding and activation of CheY by phosphorylation may be related. Our results indicate that the conformational change and charge interactions brought about by the binding of a metal ion at the active site are required for CheY to interact with a phosphate. These studies also demonstrate the utility of spin-label-induced relaxation in conjunction with two-dimensional-n.m.r. measurements for exploring ligand-binding sites.

Adenine↗