Biomedical subjects

A F Bell

Publications and source records attributed to A F Bell.

At least 19 recordsLinked to original sources

PubMed2003

Vibrationally resolved photoabsorption spectroscopy of red fluorescent protein chromophore anions.

Photoabsorption studies of red fluorescent protein chromophore anions have been performed at the ELISA electrostatic heavy-ion storage ring. The broad absorption band due to electronic excitation of the chromophores is tuned to a longer wavelength (redshifted) by extending the electronic conjugation of the molecule. A clear vibrational progression is resolved with E(vib) approximately 380 and 520 cm(-1) for two different forms of the chromophore. The vibrational modes correspond to collective motions of the entire molecular structure. It is argued that the excited electronic state has an equilibrium configuration far from that of the electronic ground state, i.e., poor Franck Condon overlap.

Absorption↗

PubMed2001

Involvement of glycine 141 in substrate activation by enoyl-CoA hydratase.

Raman spectroscopy has been used to investigate the structure of a substrate analogue, hexadienoyl-CoA (HD-CoA), bound to wild-type enoyl-CoA hydratase and G141P, a mutant in which a hydrogen bond to the substrate carbonyl has been removed. Raman spectra of isotopically labeled HD-CoAs, together with normal mode calculations, confirm the selective ground-state polarization of the enone fragment previously suggested to occur on binding to the wild-type enzyme [Tonge, P. J., Anderson, V. E., Fausto, R., Kim, M., Pusztai-Carey, M., and Carey, P. R. (1995) Biospectroscopy 1, 387-394]. In addition, Raman spectra of HD-CoA bound to the G141P mutant enzyme demonstrate that the hydrogen bond between the G141 amide NH group and the substrate carbonyl is critical for polarization and activity. Replacement of G141 with proline results in an approximately 10(6)-fold decrease in k(cat) and eliminates the ability of the enzyme to polarize the substrate analogue. As G141 is part of a consensus sequence in the enoyl-CoA hydratase superfamily, the results presented here provide direct evidence for the importance of the oxyanion hole in the reactions catalyzed by other family members.

Acyl Coenzyme A↗

PubMed2001

Active site heterogeneity in dimethyl sulfoxide reductase from Rhodobacter capsulatus revealed by Raman spectroscopy.

Raman spectroscopy has been used to investigate the structure of the molybdenum cofactor in DMSO reductase from Rhodobacter capsulatus. Three oxidized forms of the enzyme, designated 'redox cycled', 'as prepared', and DMSOR(mod)D, have been studied using 752 nm laser excitation. In addition, two reduced forms of DMSO reductase, prepared either anaerobically using DMS or using dithionite, have been characterized. The 'redox cycled' form has a single band in the Mo=O stretching region at 865 cm(-1) consistent with other studies. This oxo ligand is found to be exchangeable directly with DMS(18)O or by redox cycling. Furthermore, deuteration experiments demonstrate that the oxo ligand in the oxidized enzyme has some hydroxo character, which is ascribed to a hydrogen bonding interaction with Trp 116. There is also evidence from the labeling studies for a modified dithiolene sulfur atom, which could be present as a sulfoxide. In addition to the 865 cm(-1) band, an extra band at 818 cm(-1) is observed in the Mo=O stretching region of the 'as prepared' enzyme which is not present in the 'redox cycled' enzyme. Based on the spectra of unlabeled and labeled DMS reduced enzyme, the band at 818 cm(-1) is assigned to the S=O stretch of a coordinated DMSO molecule. The DMSOR(mod)D form, identified by its characteristic Raman spectrum, is also present in the 'as prepared' enzyme preparation but not after redox cycling. The complex mixture of forms identified in the 'as prepared' enzyme reveals a substantial degree of active site heterogeneity in DMSO reductase.

Bacterial Proteins↗

PubMed2000

Medium-chain acyl-coenzyme A dehydrogenase bound to a product analogue, hexadienoyl-coenzyme A: effects on reduction potential, pK(a), and polarization.

2,4-Hexadienoyl-coenzyme A (HD-CoA) has been used to investigate the redox and ionization properties of medium-chain acyl-CoA dehydrogenase (MCAD) from pig kidney. HD-CoA is a thermodynamically stabilized product analogue that binds tightly to oxidized MCAD (K(dox) = 3.5 +/- 0.1 microM, pH 7.6) and elicits a redox potential shift that is 78% of that observed with the natural substrate/product couple [Lenn, N. D., Stankovich, M. T., and Liu, H. (1990) Biochemistry 29, 3709-3715]. The midpoint potential of the MCAD.HD-CoA complex exhibits a pH dependence that is consistent with the redox-linked ionization of two key glutamic acids as well as the flavin adenine dinucleotide (FAD) cofactor. The estimated ionization constants for Glu376-COOH (pK(a,ox) approximately 9.3) and Glu99-COOH (pK(a,ox) approximately 7.4) in the oxidized MCAD.HD-CoA complex indicate that while binding of the C(6) analogue makes Glu376 a stronger catalytic base (pK(a,ox) approximately 6.5, free MCAD), it has little effect on the pK of Glu99 (pK(a,ox) approximately 7.5, free MCAD) [Mancini-Samuelson, G. J., Kieweg, V., Sabaj, K. M., Ghisla, S., and Stankovich, M. T. (1998) Biochemistry 37, 14605-14612]. This finding is in agreement with the apparent pK of 9.2 determined for Glu376 in the human MCAD.4-thia-octenoyl-CoA complex [Rudik, I., Ghisla, S., and Thorpe, C. (1998) Biochemistry 37, 8437-8445]. The pK(a)s estimated for Glu376 and Glu99 in the reduced pig kidney MCAD.HD-CoA complex, 9.8 and 8.6, respectively, suggest that both of these residues remain protonated in the charge-transfer complex under physiological conditions. Polarization of HD-CoA in the enzyme active site may contribute to the observed pK(a) and redox potential shifts. Consequently, the electronic structures of the product analogue in its free and MCAD-bound forms have been characterized by Raman difference spectroscopy. Binding to either the oxidized or reduced enzyme results in localized pi-electron polarization of the hexadienoyl C(1)=O and C(2)=C(3) bonds. The C(4)=C(5) bond, in contrast, is relatively unaffected by binding. These results suggest that, upon binding to MCAD, HD-CoA is selectively polarized such that partial positive charge develops at the C(3)-H region of the ligand, regardless of the oxidation state of the enzyme.

Acyl Coenzyme A↗

PubMed2000

Probing the ground state structure of the green fluorescent protein chromophore using Raman spectroscopy.

We present Raman spectra, obtained using 752 nm excitation, on wild-type GFP and the S65T mutant of this intrinsically fluorescent protein together with data on a model chromophore, ethyl 4-(4-hydroxyphenyl)methylidene-2-methyl-5-oxoimidazolacetate . In the pH range 1-14, the model compound has two macroscopic pK(a)s of 1.8 and 8.2 attributed to ionization of the imidazolinone ring nitrogen and the phenolic hydroxyl group, respectively. Comparison of the model chromophore with the chromophore in wild-type GFP and the S65T mutant reveals that the cationic form, with both the imidazolinone ring nitrogen and the phenolic oxygen protonated, is not present in these particular GFP proteins. Our results do not provide any evidence for the zwitterionic form of the chromophore, with the phenolic group deprotonated and the imidazolinone ring nitrogen protonated, being present in the GFP proteins. In addition, since the position of the Raman bands is a property exclusively of the ground state structure, the data enable us to investigate how protein-chromophore interactions affect the ground state structure of the chromophore without contributions from excited state effects. It is found that the ground state structure of the anionic form of the chromophore, which is most relevant to the fluorescent properties, is strongly dependent on the chromophore environment whereas the neutral form seems to be insensitive. A linear correlation between the absorption properties and the ground state structure is demonstrated by plotting the absorption maxima versus the wavenumber of a Raman band found in the range 1610-1655 cm(-1).

Acetates↗

PubMed2000

Solution structure and dynamics of biomolecules from Raman optical activity.

Raman optical activity (ROA) measures vibrational optical activity by means of a small difference in the intensity of Raman scattering from chiral molecules in right and left circularly polarized incident laser light. The ROA spectra of a wide range of biomolecules in aqueous solution can now be measured routinely. Because of its sensitivity to the chiral elements of biomolecular structure, ROA provides new information about solution structure and dynamics complementary to that supplied by conventional spectroscopic techniques. This article provides a brief introduction to the theory and practice of ROA spectroscopy followed by a review of recent ROA results on polypeptides, proteins, carbohydrates, nucleic acids and viruses which illustrate how new insight into current problems of structure, folding and function may be obtained from ROA studies.

Animals↗

PubMed1999

Raman optical activity of filamentous bacteriophages: hydration of alpha-helices.

We report the first observations of vibrational Raman optical activity (ROA) on intact viruses. Specifically, ROA spectra of the filamentous bacteriophages Pf1, M13 and IKe in aqueous solution were measured in the range approximately 600-1800 cm-1. On account of its ability to probe directly the chiral elements of biomolecular structure, ROA has provided a new perspective on the solution structures of these well-studied systems. The ROA spectra of all three are dominated by signatures of helical elements in the major coat proteins, as expected from pre-existing data. The helical elements generate strong sharp positive ROA bands at approximately 1300 and 1342 cm-1in H2O solution, but in2H2O solution the approximately 1342 cm-1bands disappear completely. The spectra are similar to those of polypeptides under conditions that produce alpha-helical conformations. Our present results, together with results from other studies, suggest that the positive approximately 1342 cm-1ROA bands are generated by a highly hydrated form of alpha-helix, and that the positive approximately 1300 cm-1bands originate in alpha-helix in a more hydrophobic environment. The presence of significant amounts of highly hydrated helical sequences accords with the known flexibility of these viruses. Differences of spectral detail for Pf1, M13 and IKe demonstrate that ROA is sensitive to subtle variations of conformation and hydration within the major coat proteins, with M13 and IKe possibly containing more non-helical structure than Pf1. The ROA spectra of Pf1 at temperatures above and below that at which a structural transition is known to occur (approximately 10 degrees C) reveal little difference in the protein conformation between the two forms, but there are indications of changes in DNA structure.

Amino Acid Sequence↗

PubMed1998

Evidence for global mobility in the premelting of a polynucleotide from temperature-dependent Raman optical activity.

The backscattered Raman and Raman optical activity (ROA) spectra of poly(rA)-poly(rU) at 20 degrees C and 45 degrees C in buffered aqueous solution between 650 and 1750 cm(-1) are reported. Although the intensity of the majority of the Raman bands increase by varying amounts as the temperature is raised in accordance with the well-known hypochromic effect, the reverse effect is found for the ROA signals which we attribute to thermal accessibility of a greater number of distinct conformations leading to cancellation of ROA signals. The difference ROA spectrum obtained by subtracting the spectrum recorded at 45 degrees C from that recorded at 20 degrees C displays a very similar sign pattern to those at both 20 degrees C and 45 degrees C throughout the spectral region examined. This indicates that the same average structure is maintained in this temperature range and that the thermal fluctuations are correlated through the bases, the glycosidic link, the sugar ring, and the phosphate backbone of both strands. These results indicate that ROA may be a useful new probe of the dynamics of nucleic acid in solution.

Poly A-U↗

PubMed1998

The effects of linear acceleration on distortion product otoacoustic emissions in human ears.

BACKGROUND: The effect of high levels of linear acceleration (G) on the cochlea has never been studied prospectively. HYPOTHESIS: Linear acceleration at high levels has no effect on the human cochlea as demonstrated by a comparison of pre- and post-exposure measurements. METHODS: There were 22 healthy volunteers who underwent exposure to up to 9 G in a military aviation training centrifuge. Prior to exposure they were screened for cochleovestibular disorders and underwent tympanometry, audiometry and distortion product otoacoustic emissions testing (DPOAE). Immediately after exposure, they underwent serial testing of each of these parameters until they returned to baseline. RESULTS: There was no significant change in tympanometry in any subject. Audiometry revealed a temporary threshold shift of 30 db at 6 kHz in one ear of a single subject. This was accompanied by a complete loss of DPOAE at the same frequency. DPOAE did not return to baseline at 2 weeks even though the audiogram had reverted to baseline by 8 d. Four other ears displayed significant losses of emissions at single frequencies without an accompanying change on the audiogram. CONCLUSIONS: This study demonstrates that accelerative stress may cause transient injury to the cochlea. The mechanism of injury due to acceleration is probably ischemia, although a purely mechanical effect on the outer hair cells cannot be precluded. These data also reinforce a growing body of evidence that demonstrates the greater sensitivity of DPOAE over psychoacoustic testing in detecting early or subclinical cochlear damage.

Acceleration↗

PubMed1998

Raman optical activity: an incisive new probe of the structure and dynamics of biomolecules.

Determination of the solution structure and function of biomolecules such as proteins, carbohydrates and nucleic acids remains at the forefront of biomedical research. Thanks to recent developments in instrumentation, biomolecules can now be studied using Raman optical activity (ROA), a novel technique which measures vibrational optical activity in chiral molecules by means of a small difference in the intensity of Raman scattering in right- and left-circularly polarized incident laser light. This article reviews recent progress in the application of ROA to the determination of the solution structure and dynamics of proteins, carbohydrates and nucleic acids.

Carbohydrate Conformation↗

PubMed1997

Measles virus infection of human T cells modulates cytokine generation and IL-2 receptor alpha chain expression.

Measles virus (MV) suppresses specific functions in cells of the immune system and causes a generalized immunosuppression by mechanisms which remain undefined. It has been previously established that mitogen-induced proliferation of peripheral blood mononuclear cells (PBMC) is suppressed by infection with MV. Our current study demonstrates that MV infection inhibits antigen-specific proliferation of T lymphocytes. The inhibition of proliferation was not due to a decrease in IL-2 production. IL-2 production in cultures of infected and uninfected antigen-specific T cells was similar. In contrast, we found that expression of the IL-2R alpha subunit was decreased in mitogen-stimulated, MV-infected PBMC and antigen-stimulated, MV-infected T lymphocytes compared to stimulated but noninfected T cells. However, the expression of the IL-2R beta subunit was not altered in MV-infected T cells. We also examined the influence of MV infection on the production of the cytokines IL-4, IL-6, IL-10, and IFN-gamma by T lymphocytes. By comparing infected versus uninfected antigen-specific T cell lines, we found that MV infection of antigen-specific activated T cells caused no substantial change in generation of IFN-gamma, IL-6, or IL-10. There was a 50% reduction in IL-4 generation following MV infection. These data indicate that the immunosuppression by acute MV infection is not associated with a generalized inhibition of cytokine production. One mechanism for the suppression of proliferation following acute MV infection may be a block in the expression of the IL-2R alpha subunit by activated T cells.

CD4-Positive T-Lymphocytes↗

PubMed1997

Antisense-mediated resistance to measles virus infection in HeLa cells.

Endogenous expression of antisense RNA in transfected cells has been explored for use in blocking cellular gene expression and for its antiviral potential. Antisense strategies were used with the goal of blocking measles virus (MV) infection. A recombinant expression plasmid was designed to produce antisense oligonucleotides targeted to the 5' end of the MV nucleocapsid protein mRNA. This construct was transfected into HeLa cells. The transfected cell line and a control cell line expressing a random RNA comprising the same nucleotides were infected with MV and assessed for viral resistance by observation of cytopathic effect (CPE); infectious virus was quantified by viral plaque assay. Both cell lines were also infected with a related paramyxovirus, mumps virus, as a specificity control. Both CPE and infectious virus were reduced by approximately 90% in the antisense-expressing line compared with that in control cells or transfectant cells expressing random RNA. There was no evidence of resistance to infection with mumps virus in any cell line.

Cytopathogenic Effect, Viral↗

PubMed1995

Barosinus pneumocele: transient visual loss due to sphenoid sinus pneumocele in a U.S. Air Force pilot.

Symptomatic sphenoid sinus pneumoceles are extremely rare. This case presented in a U.S. Air Force aviator who had recurrent left-sided visual loss during ascent in jet aircraft. Computed tomography showed a dehiscent optic nerve within the left sphenoid sinus. Surgical correction included removing a mucosal polyp from the sinus ostium. The aviator has since flown 70 sorties without recurrence of symptoms. This entity is named barosinus pneumocele.

Adult↗

PubMed1995

Traumatic vertebral artery occlusion in an aviator: case report and update on diagnostic technologies.

Traumatic vertebral artery injuries are relatively rare. Until recently, insufficient neurodiagnostic technology and a lack of normative data for the population of USAF aviators prevented recommending flying waivers following such vascular injuries. We report the case of an aviator who experienced a traumatic right vertebral artery occlusion with distal embolization. Timely access to a state-of-the-art neurodiagnostic evaluation, which included time-of-flight magnetic resonance angiography (MRA), and standard contrast angiography, provided anatomic imaging at the time of injury and during the recuperative period. Sophisticated neurologic testing protocols at the Armstrong Laboratory's Aeromedical Clinical Sciences Division, Brooks Air Force Base, TX, assessed in-depth this individual's functional status. Consult Service evaluators then compared his performance data with current data in the Head Injury Study in Aviators (HISA) data base. MRA, in comparison with standard contrast angiography, demonstrated anatomic stability of his vascular and neurologic lesions. He demonstrated a functional capacity consistent with normal neurologic functioning in aviators which permitted recommending an occupational (flying) waiver. This case exemplified application of sophisticated ACS neurologic testing protocols to aeromedical evaluations and the use of MRA technology as a vascular screening tool during medical followup.

Adult↗

PubMed1994

Vibrational Raman optical activity of glycoproteins.

This paper reports the first vibrational Raman optical activity (ROA) spectrum of a glycoprotein. The sample, orosomucoid (alpha 1-acid glycoprotein), shows ROA bands characteristic of a high beta-sheet content together with new bands which could be specific for the carbohydrate and its association with the protein. Our results suggest that ROA spectra of intact glycoproteins may contain information about both protein and carbohydrate conformation and the mutual influence on each other's stability and conformation.

Carbohydrate Conformation↗

PubMed1994

Analysis of pursuit tracking eye movements in pilots and nonfliers.

Pursuit tracking eye movements were recorded and analyzed from a group of Air Force pilots and a group of nonflying Air Force members. The tracking performance of the pilots was compared to the performance of the nonfliers. Subjects tracked a small spot of light moving sinusoidally in the horizontal plane at frequencies ranging from 0.2 to 1.0 Hz at a peak-to-peak amplitude of 40 degrees. Maximum target velocities ranged from 25 to 126 degrees/s. An adaptive nonlinear digital filter was used to separate the total tracking response (TTR) into smooth pursuit (SP) and saccadic (SA) components. Frequency domain analysis was used to relate the tracking components to the target movement. There were no statistically significant differences in tracking performance between the pilots and nonfliers. When tracking the 0.2 and 0.4 Hz targets, the TTR consisted principally of SP tracking with SA tracking representing less than 11% of the TTR. As the target velocity increased, the TTR remained adequate but the proportion of SP tracking decreased while the SA tracking increased. Over one-half of the TTR is contributed by the SA system when tracking the 1.0 Hz targets for both the pilot and nonpilot groups.

Adult↗

PubMed1994

The application of smooth pursuit eye movement analysis to clinical medicine.

Pursuit tracking eye movements were analyzed from selected patients with neurological injuries and compared to the responses of 20 normal subjects. The patients/subjects tracked a small spot of light moving sinusoidally in the horizontal plane at a frequency of 0.4 Hz and a peak-to-peak amplitude of 40 degrees. Eye-movement responses were separated into a smooth-pursuit component and a saccadic component. The smooth-pursuit component was analyzed by calculating the gain, phase, and asymmetry. The saccadic component was quantified by calculating the percentage of the total tracking movement contributed by the saccadic system. The patients with smooth-pursuit impairment exhibited a higher percentage of saccadic tracking and a lower smooth pursuit gain compared to the normal subjects. One patient with a unilateral lesion exhibited significant asymmetry in the smooth-pursuit component. In this case, the direction of the asymmetry indicated the side of the lesion.

Adult↗

PubMed1993

Linear systems analysis of the vestibulo-ocular reflex: clinical applications.

We used whole-body angular acceleration stimuli to estimate the transfer function of the vestibuloocular reflex in 20 normal subjects and several patients. Eye movements evoked by the stimuli were recorded and an adaptive nonlinear digital filter was used to extract the compensatory component of the eye-movement response. Frequency domain analysis of the stimulus and the compensatory component of the response was used to estimate the transfer function. The transfer function was estimated at 6 discrete frequencies (0.01 to 0.32 Hz by octaves) using individual sine-wave stimuli. Data from several patients with known lesions affecting the vestibular system were compared to the normal responses. Patients with a unilateral vestibular loss demonstrated low gains at frequencies below 0.02 Hz and large phase shifts below 0.32 Hz, but had normal responses at 0.32 Hz. Some of these same patients also exhibited a significant directional asymmetry in their responses.

Adult↗