Biomedical subjects

M K Johnson

Publications and source records attributed to M K Johnson.

At least 37 records · Page 2Linked to original sources

PubMed2000

Modular organization and identification of a mononuclear iron-binding site within the NifU protein.

The NifS and NifU nitrogen fixation-specific gene products are required for the full activation of both the Fe-protein and MoFe-protein of nitrogenase from Azotobacter vinelandii. Because the two nitrogenase component proteins both require the assembly of [Fe-S]-containing clusters for their activation, it has been suggested that NifS and NifU could have complementary functions in the mobilization of sulfur and iron necessary for nitrogenase-specific [Fe-S] cluster assembly. The NifS protein has been shown to have cysteine desulfurase activity and can be used to supply sulfide for the in vitro catalytic formation of [Fe-S] clusters. The NifU protein was previously purified and shown to be a homodimer with a [2Fe-2S] cluster in each subunit. In the present work, primary sequence comparisons, amino acid substitution experiments, and optical and resonance Raman spectroscopic characterization of recombinantly produced NifU and NifU fragments are used to show that NifU has a modular structure. One module is contained in approximately the N-terminal third of NifU and is shown to provide a labile rubredoxin-like ferric-binding site. Cysteine residues Cys35, Cys62, and Cys106 are necessary for binding iron in the rubredoxin-like mode and visible extinction coefficients indicate that up to one ferric ion can be bound per NifU monomer. The second module is contained in approximately the C-terminal half of NifU and provides the [2Fe-2S] cluster-binding site. Cysteine residues Cys137, Cys139, Cys172, and Cys175 provide ligands to the [2Fe-2S] cluster. The cysteines involved in ligating the mononuclear Fe in the rubredoxin-like site and those that provide the [2Fe-2S] cluster ligands are all required for the full physiological function of NifU. The only two other cysteines contained within NifU, Cys272 and Cys275, are not necessary for iron binding at either site, nor are they required for the full physiological function of NifU. The results provide the basis for a model where iron bound in labile rubredoxin-like sites within NifU is used for [Fe-S] cluster formation. The [2Fe-2S] clusters contained within NifU are proposed to have a redox function involving the release of Fe from bacterioferritin and/or the release of Fe or an [Fe-S] cluster precursor from the rubredoxin-like binding site.

Amino Acid Sequence↗

PubMed2000

fMRI evidence of age-related hippocampal dysfunction in feature binding in working memory.

Richly detailed memories for particular events depend on processes that bind individual features of experience together. Previous cognitive behavioral research indicates that older adults have more difficulty than young adults in conditions requiring feature binding. We used functional magnetic resonance imaging (fMRI) during a working memory task to identify neural substrates of this age-related deficit in feature binding. For young, but not older, adults there was greater activation in left anterior hippocampus on combination trials (remember objects together with their locations) than on trials in which participants were told to remember only which objects or only which locations occurred. The results provide neuroimaging evidence for an age-related hippocampal dysfunction in feature binding in working memory.

Adult↗

PubMed2000

Cross-modal source monitoring confusions between perceived and imagined events.

Two experiments tested the prediction based on the source monitoring framework that imagination is most likely to lead to false memories when related perceived events have occurred. Consistent with this, people were more likely to falsely remember seeing events when the events had been both imagined as seen and actually heard than when they were just heard, just visually imagined, or imagined both visually and auditorily. Furthermore, when people considered potential sources for memories or more carefully evaluated features of remembered events, source errors were reduced. On average, misattributed ("false") memories differed in phenomenal qualities from true memories. Taken together, these findings show that as different qualities of mental experience flexibly enter into source attributions, qualities derived from related perceptual events are particularly likely to lead to false claims that imagined events were seen, even when the event involves a primary modality (auditory) different from the target event (visual).

Adult↗

PubMed2000

Aging and reflective processes of working memory: binding and test load deficits.

It was hypothesized that age-related deficits in episodic memory for feature combinations (e.g., B. L. Chalfonte & M. K. Johnson, 1996) signal, in part, decrements in the efficacy of reflective component processes (e.g., M. K. Johnson, 1992) that support the short-term maintenance and manipulation of information during encoding (e.g., F. 1. M. Craik. R. G. Morris. & M. L. Gick, 1990; T. A. Salthouse, 1990). Consistent with this, age-related binding deficits in a working memory task were found in 2 experiments. Evidence for an age-related test load deficit was also found: Older adults had greater difficulty than young adults when tested on 2 features rather than 1, even when binding was not required. Thus, disruption of source memory in older adults may involve deficits in both encoding processes (binding deficits) and monitoring processes (difficulty accessing multiple features, evaluating them, or both).

Adolescent↗

PubMed2000

Choice-supportive source monitoring: do our decisions seem better to us as we age?

Participants were given several 2-option choices and then asked to review how they felt about their decisions, to review the details of their decisions, or to do an unrelated task. When later asked to attribute features to the previous options, in each condition older adults (64-83 years) attributed significantly more positive and fewer negative features to their chosen options than to foregone options. Younger adults' (18-22 years) attributions were as choice-supportive as those of older adults in the affective review condition but were less so in the other conditions. The age difference was present even when older and younger adults were equated for source identification and recognition accuracy. This study suggests that as people age, their tendency to distort memory in favor of the options they chose increases. In addition, it suggests that affectively reviewing choices increases younger adults' tendency toward choice-supportive memory.

Adolescent↗

PubMed2000

Misremembrance of options past: source monitoring and choice.

This study reveals that when remembering past decisions, people engage in choice-supportive memory distortion. When asked to make memory attributions of options' features, participants made source-monitoring errors that supported their decisions. They tended to attribute, both correctly and incorrectly, more positive features to the option they had selected than to its competitor. In addition, they sometimes attributed, both correctly and incorrectly, more negative features to the nonselected option. This pattern of distortion may be beneficial to people's general well-being, reducing regret for options not taken. At the same time, it is problematic for memory accuracy, for accountability, and for learning from past experience.

Adult↗

PubMed2000

Identification of acylpeptide hydrolase as a sensitive site for reaction with organophosphorus compounds and a potential target for cognitive enhancing drugs.

We describe here the purification and identification of a previously unrecognized target for organophosphorus compounds. The target, acylpeptide hydrolase, was isolated as a tritiated-diisopropylfluorophosphate-reactive protein from porcine brain and purified to homogeneity using a combination of ion-exchange and gel-filtration chromatography. Biochemical characterization and internal sequence analysis confirmed identity. Acylpeptide hydrolase was found to be potently inhibited by the organophosphorus compounds chlorpyrifosmethyl oxon, dichlorvos, and diisopropylfluorophosphate (20-min IC(50) values of 18.3 +/- 2.0, 118.7 +/- 9.7, and 22.5 +/- 1.2 nM, respectively). The in vitro sensitivity of acylpeptide hydrolase toward these compounds is between six and ten times greater than that of acetylcholinesterase (AChE), making it a target of pharmacological and toxicological significance. We show that, in vivo, acylpeptide hydrolase is significantly more sensitive than AChE to inhibition by dichlorvos and that the inhibition is more prolonged after a single dose of inhibitor. Furthermore, using dichlorvos as a progressive inhibitor, it was possible to show that acylpeptide hydrolase is the only enzyme in the brain capable of hydrolyzing the substrate N-acetyl-alanyl-p-nitroanilide. A concentration of 154 +/- 27 pmol of acylpeptide hydrolase/gram of fresh rat brain was also deduced by specific labeling with tritiated-diisopropylfluorophosphate. We also suggest that, by comparison of structure-activity relationships, acylpeptide hydrolase may be the target for the cognitive-enhancing effects of certain organophosphorus compounds. Acylpeptide hydrolase cleaves N(alpha)-acylated amino acids from small peptides and may be involved in regulation of neuropeptide turnover, which provides a new and plausible mechanism for its proposed cognitive enhancement effect.

Animals↗

PubMed2000

Early discharge for patients with exacerbations of chronic obstructive pulmonary disease: a randomized controlled trial.

BACKGROUND: We have previously reported the use of a hospital based respiratory nurse service (Acute Respiratory Assessment Service, ARAS) to support home treatment of patients with exacerbations of chronic obstructive pulmonary disease (COPD). A controlled trial was undertaken to compare early discharge with home treatment supported by respiratory nurses with conventional hospital management of patients admitted with exacerbations of COPD. METHODS: Patients with COPD admitted as emergencies were identified the next working day. They were eligible for inclusion in the study if the differential diagnosis included an exacerbation of COPD, but were excluded if other medical conditions or acidotic respiratory failure required inpatient investigation or management. Of 360 patients reviewed, 209 were being assessed for other active medical problems and were excluded, 33 potential participants were already involved in research studies and so were ineligible, and 37 did not wish to participate in the study. Eighty one patients were randomised to receive conventional inpatient care (n=40) or to planned early discharge the next working day (n=41). Those discharged early continued treatment at home under the supervision of specialist respiratory nurses. Outcome measures were readmission, additional hospital days, and deaths within 60 days of initial admission. Process measures included number of visits, duration of follow up by the respiratory nurse, and additional treatment provided to support early discharge. RESULTS: On an intention to treat basis, a policy of early discharge reduced inpatient stay from a mean of 6.1 (range 1-13) days with conventional management to 3.2 (1-16) days with an early discharge policy. Twelve patients (30% conventional management, 29.3% early discharge) were readmitted in each group giving a mean difference in readmission of 0.7% (95% CI of the difference -19.2 to 20.6). In the conventional management group readmitted patients spent a mean of 8.75 additional days in hospital compared with 7.83 days in the early discharge group, giving a mean difference of 0.92 days (95% CI of the difference -6.5 to 8.3). There were two deaths (5%) in the conventional management group and one (2.4%) in the early discharge group, a mean difference of 2.6% (95% CI of the difference -5.7 to 10.8). CONCLUSIONS: Patients with acute exacerbations of COPD uncomplicated by acidotic respiratory failure or other medical problems can be discharged home earlier than is current practice with support by visiting respiratory nurses. No difference was found in the subsequent need for readmission.

Aged↗

PubMed2000

Large lung bullae in marijuana smokers.

The case histories are presented of four men with multiple large upper zone lung bullae but otherwise relatively preserved lung parenchyma. Each had a history of significant exposure to marijuana. In three of the four cases the tobacco smoking load had been relatively small, suggesting a possible causal role for marijuana in the pathogenesis of this unusual pattern of bullous emphysema.

Adult↗

PubMed2000

Liquidity-related plan asset issues.

By about 2025, most baby boomers will have retired, which will put a tremendous strain on public sector pension plans. Many will experience negative cash flows, and liquidity will be an increasing concern. Asset/liability studies can help measure the effect of this risk on system funding and contribution requirements, resulting in more informed asset allocation choices and benefit policies.

Aged↗

PubMed1999

Potent antioxidant activity of a dithiocarbamate-related compound from a marine hydroid.

Recently, we discovered a novel class of natural products, named the tridentatols, in a marine hydroid. Close examination of their molecular structures suggested that they may have antioxidant activity. This observation prompted us to evaluate in vitro the capacity of one of these tridentatols, viz. tridentatol A, to inhibit lipid peroxidation using human low density lipoprotein (LDL) as an experimental model. LDL was incubated with 5 microM cupric chloride (Cu2+) in the absence and presence of tridentatol A or a reference antioxidant standard, i.e. vitamin E. The onset of rapid formation of conjugated lipid hydroperoxides was delayed in a concentration-dependent manner by tridentatol A. More specifically, LDL incubated with Cu2+ had a lag-phase time (the elapsed time before the onset of rapid formation of conjugated lipid hydroperoxides) of 150 min. However, when 0.5 microM tridentatol A was present during incubation, the lag phase time was extended to 225 min. With 1 microM tridentatol A, the lag phase time was 300 min. The same concentrations of vitamin E produced noticeably lower lag phase times. Thus, compared with vitamin E, tridentatol A better protected against the formation of conjugated lipid hydroperoxides in LDL. Direct colorimetric measurements of both lipid hydroperoxides and thiobarbituric acid-reactive substances confirmed the greater potency of tridentatol A relative to vitamin E. Furthermore, tridentatol A negated the Cu2+-induced increase in electrophoretic mobility of LDL to a greater extent than vitamin E. In conclusion, tridentatol A is a powerful antioxidant against lipid peroxidation of LDL and is significantly more potent than vitamin E in this regard.

Animals↗

PubMed1999

Probing the heme axial ligation in the CO-sensing CooA protein with magnetic circular dichroism spectroscopy.

The combination of UV/visible/near-IR variable-temperature magnetic circular dichroism (VTMCD) and EPR spectroscopies has been used to investigate the spin states and axial ligation of the heme group in oxidized, reduced, and CO-bound reduced forms of the Rhodospirillum rubrum CO oxidation transcriptional activator protein (CooA) and its H77Y and C75S variants. The energy of the porphyrin(pi)-to-Fe(III) charge-transfer band (8930 cm(-)(1)) and the presence of cysteinate S-to-Fe(III) charge-transfer bands between 600 and 700 nm confirm cysteinate axial ligation to the low-spin Fe(III) hemes in oxidized wild-type and H77Y CooA. In contrast, the major component in the oxidized C75S variant is shown to be a low-spin Fe(III) heme with bis-histidine or histidine/amine axial ligation on the basis of the energy of the porphyrin(pi)-to-Fe(III) charge-transfer band (6240 cm(-)(1)) and the anisotropy of the EPR signal, g = 3.23, approximately 2.06, approximately 1.14. These results confirm Cys75 as the cysteinyl axial ligand in oxidized CooA, indicate that it is replaced as an axial ligand by a histidine in the C75S variant, and reveal the presence of a hitherto unidentified histidine or neutral nitrogen ligand trans to Cys75 in wild-type CooA. Evidence for a Cys75-to-His77 axial ligand switch on reduction of CooA comes from VTMCD studies of the reduced proteins. The VTMCD spectra of reduced wild-type and C75S CooA are dominated by bands characteristic of bis-histidine low-spin Fe(II) hemes, whereas the reduced H77Y variant is predominantly high-spin with MCD characteristics typical of a five-coordinate, histidine-ligated ferrous heme. VTMCD studies show that the CO-bound reduced forms of wild-type, H77Y, and C75S contain low-spin Fe(II) hemes and that the Fe-CO bonds can be photolytically cleaved at temperatures <50 K. Strong evidence that CO binding to the heme group in reduced CooA occurs with displacement of His77 comes from the VTMCD spectra of the low-temperature photoproducts of CO-bound reduced forms of wild-type, H77Y, and C75S CooA. The spectra are almost identical to each other and closely correspond to those of the low-temperature photoproducts of well characterized CO-bound ferrous hemes with His/CO axial ligation.

Bacterial Proteins↗

PubMed1999

Effects of mutations in aspartate 14 on the spectroscopic properties of the [Fe3S4]+,0 clusters in Pyrococcus furiosus ferredoxin.

The properties of [Fe(3)S(4)](+,0) clusters in wild-type and mutant forms of Pf Fd with Asp, Ser, Cys, Val, His, Asn, and Tyr residues occupying position 14, i.e., proximal to the three micro(2)-S atoms of the cluster, have been investigated by the combination of EPR, variable-temperature magnetic circular dichroism (VTMCD), and resonance Raman (RR) spectroscopies. Two distinct types of [Fe(3)S(4)] clusters are identified on the basis of the breadth of the S = (1)/(2) [Fe(3)S(4)](+) EPR resonances and the marked differences in the VTMCD spectra of the S = 2 [Fe(3)S(4)](0) clusters. On the basis of the available NMR data for [Fe(3)S(4)](+, 0) clusters in ferredoxins, the distinctive properties of these two types of [Fe(3)S(4)] clusters are interpreted in terms of different locations of the more strongly coupled pair of irons in the oxidized clusters and the valence-delocalized pair in the reduced clusters. Near-IR VTMCD measurements indicate the presence of S = (9)/(2) valence-delocalized pairs in both types of [Fe(3)S(4)](0) clusters, and the spin-dependent delocalization energies associated with the Fe-Fe interactions were determined to be approximately 4300 cm(-)(1) in both cases. We conclude that the nature of the residue at position 14 in Pyrococcus furiosus ferredoxin is an important determinant of the location of the reducible pair of irons in a [Fe(3)S(4)](+,0) cluster, and the redox properties of the wild-type and mutant ferredoxins are discussed in light of these new results.

Aspartic Acid↗

PubMed1999

Effect of serinate ligation at each of the iron sites of the [Fe4S4] cluster of Pyrococcus furiosus ferredoxin on the redox, spectroscopic, and biological properties.

Pyrococcus furiosus ferredoxin (Fd) contains a single [Fe(4)S(4)] cluster coordinated by three cysteine (at positions 11, 17, and 56) and one aspartate ligand (at position 14). In this study, the spectroscopic, redox, and functional consequences of D14C, D14C/C11S, D14S, D14C/C17S, and D14C/C56S mutations have been investigated. The four serine variants each contain a potential cluster coordination sphere of one serine and three cysteine residues, with serine ligation at each of the four Fe sites of the [Fe(4)S(4)] cluster. All five variants were expressed in Escherichia coli, and each contained a [Fe(4)S(4)](2+,+) cluster as shown by UV-visible absorption and resonance Raman studies of the oxidized protein and EPR and variable-temperature magnetic circular dichroism (VTMCD) studies of the as-prepared, dithionite-reduced protein. Changes in both the absorption and resonance Raman spectra are consistent with changing from complete cysteinyl cluster ligation in the D14C variant to three cysteines and one oxygenic ligand in each of the four serine variants. EPR and VTMCD studies show distinctive ground and excited state properties for the paramagnetic [Fe(4)S(4)](+) centers in each of these variant proteins, with the D14C and D14C/C11S variants having homogeneous S = (1)/(2) ground states and the D14S, D14C/C17S, and D14C/C56S variants having mixed-spin, S = (1)/(2) and (3)/(2) ground states. The midpoint potentials (pH 7.0, 23 degrees C) of the D14C/C11S and D14C/C17S variants were unchanged compared to that of the D14C variant (E(m) = -427 mV) within experimental error, but the potentials of D14C/C56S and D14S variants were more negative by 49 and 78 mV, respectively. Since the VTMCD spectra indicate the presence of a valence-delocalized Fe(2. 5+)Fe(2.5+) pair in all five variants, the midpoint potentials are interpreted in terms of Cys11 and Cys17 ligating the nonreducible valence-delocalized pair in D14C. Only the D14S variant exhibited a pH-dependent redox potential over the range of 3.5-10, and this is attributed to protonation of the serinate ligand to the reduced cluster (pK(a) = 4.75). All five variants had similar K(m) and V(m) values in a coupled assay in which Fd was reduced by pyruvate ferredoxin oxidoreductase (POR) and oxidized by ferredoxin NADP oxidoreductase (FNOR), both purified from P. furiosus. Hence, the mode of ligation at each Fe atom in the [Fe(4)S(4)] cluster appears to have little effect on the interaction and the electron transfer between Fd and FNOR.

Amino Acid Sequence↗

PubMed1999

A pure S = 3/2 [Fe4S4]+ cluster in the A33Y variant of Pyrococcus furiosus ferredoxin.

The properties of the [4Fe-4S]2+/+ cluster in wild-type and the A33Y variant of Pyrococcus furiosus ferredoxin have been investigated by the combination of EPR, variable-temperature magnetic circular dichroism (VTMCD) and resonance Raman (RR) spectroscopies. The A33Y variant involves the replacement of an alanine whose alpha-C is less than 4 A from one of the cluster iron atoms by a tyrosine residue. Although the spectroscopic results give no indication of tyrosyl cluster ligation, the presence of a tyrosine residue in close proximity to the cluster results in a 38-mV decrease in the midpoint potential of the [4Fe-4S]2+/+ couple and has a marked effect on the ground state properties of the reduced cluster. The mixed spin [4Fe-4S]+ cluster in the wild-type protein, 80% S = 3/2 (E/D = 0.22, D = +3.3 cm(-1)) and 20% S = 1/2 (g = 2.10, 1.87, 1.80), is converted into a homogeneous S = 3/2 (E/D = 0.30, D = -0.7 cm(-1)) form in the A33Y variant. As the first example of a pure S = 3/2 [4Fe-4S]+ cluster in a ferredoxin, this variant affords the opportunity for detailed characterization of the excited electronic properties via VTMCD studies and demonstrates that the protein environment can play a crucial role in determining the ground state properties of [4Fe-4S]+ clusters.

Circular Dichroism↗

PubMed1999

Mutagenesis studies of the FeSII protein of Azotobacter vinelandii: roles of histidine and lysine residues in the protection of nitrogenase from oxygen damage.

The Azotobacter FeSII protein, also known as the Shethna protein, forms a protective complex with nitrogenase during periods when nitrogenase is exposed to oxygen. One possible mechanism for its action is an oxidation state-dependent conformational interaction with nitrogenase whereby the FeSII protein dissociates from the MoFe and Fe proteins of nitrogenase under reducing conditions. Herein we report the construction and characterization of five site-directed mutants of the FeSII protein (H12Q, H55Q, K14A, K15A, and the double mutant K14A/K15A) which were individually purified after being individually overexpressed in Escherichia coli. These mutant FeSII proteins maintain native-like assembly and orientation of the 2Fe-2S center on the basis of EPR and NMR spectroscopic characterization and their redox midpoint potentials, which are within 25 mV of that of the wild type protein. The abilities of the individual mutant proteins to protect nitrogenase were assessed by determining the remaining nitrogenase activities after adding each pure version back to extracts from an FeSII deletion strain, and then exposing the mixture to oxygen. In these assays, the H12Q mutant functioned as well as the wild type protein. However, mutation of His55, a few residues away from a cluster-liganding cysteine, results in much less efficient protection of nitrogenase. These results are consistent with pH titrations in both oxidation states, which show that His12 is insensitive to 2Fe-2S cluster oxidation state. His55's pK is weakly responsive to oxidation state, and the pK increase of 0. 16 pH unit upon 2Fe-2S cluster oxidation is indicative of ionization of another group between His55 and the 2Fe-2S cluster, which could modulate the FeSII protein's affinity for nitrogenase in a redox state-dependent manner. Both K14A and K15A mutant FeSII proteins partially lost their ability to protect nitrogenase, but the lysine double mutant lost almost all its protective ability. The nitrogenase component proteins in an Azotobacter strain bearing the double lysine mutation (in the chromosome) were degraded much more rapidly in vivo than those in the wild type strain under carbon substrate-limited conditions. These results indicate that the two lysines may have an important role in FeSII function, perhaps in the initial steps of recognizing the nitrogenase component proteins.

Azotobacter vinelandii↗

PubMed1999

The Met243 sulfonium ion linkage is responsible for the anomalous magnetic circular dichroism and optical spectral properties of myeloperoxidase.

The heme group of myeloperoxidase shows anomalous optical properties, and the enzyme possesses the unique ability to catalyze the oxidation of chloride. However, the nature of the covalently bound heme macrocycle has been difficult to identify. In this work, the electronic and magnetic properties of the heme groups in oxidized and reduced forms of wild-type and Met243Thr mutant myeloperoxidase and wild-type lactoperoxidase have been investigated using variable-temperature (1.6-273 K) magnetic circular dichroism (MCD) spectroscopy along with parallel optical absorption and electron paramagnetic resonance studies. The results provide assessment of the spin state mixtures of the oxidized and reduced samples at ambient and liquid helium temperatures and show that the anomalous MCD properties of myeloperoxidase, e.g. red-shifted and inverted signs for bands in the high-spin ferric and low-spin ferrous forms compared to other heme peroxidases and heme proteins in general, are a direct consequence of a novel electron-withdrawing sulfonium ion heme linkage involving Met243.

Circular Dichroism↗

PubMed1999

A phenolic antioxidant from the freshwater orchid, Habenaria repens.

Recently, an unusual compound named habenariol was isolated from the freshwater orchid, Habenaria repens. Its phenolic structure suggested that habenariol should have substantial antioxidant activity. This possibility was investigated by evaluating the capacity of habenariol to inhibit copper-induced lipid peroxidation of human low density lipoprotein (LDL), a popular experimental model. LDL was incubated with 5 microM cupric chloride in the presence and absence of habenariol or a positive control, viz., alpha-tocopherol. Both kinetic and end-point spectrophotometric assays were used to determine extent of lipid peroxidation of LDL. In the kinetic assay, the time elapsing before the onset of rapid formation of conjugated lipid hydroperoxides in LDL (marked by a sharp increase in UV absorbance) was prolonged by habenariol, indicative of an antioxidant effect. In the end-point assay, direct colorimetric measurement confirmed habenariol's ability to inhibit formation of lipid hydroperoxides. However, in both assays, habenariol was less potent than alpha-tocopherol in inhibiting lipid peroxidation of LDL.

Antioxidants↗