Biomedical subjects

M K Johnson

Publications and source records attributed to M K Johnson.

At least 91 records · Page 5Linked to original sources

PubMed1996

Spatial location memory in amnesia: binding item and location information under incidental and intentional encoding conditions.

Items located within an array were presented to alcoholic Korsakoff and nonalcoholic mixed-etiology amnesics and to alcoholic and normal controls. Recognition memory for the locations of items was tested after incidental and intentional encoding. When equated on item recognition, neither Korsakoff amnesics nor alcoholic controls benefited from intentional, relative to incidental, encoding instructions. Furthermore, Korsakoff amnesics showed neither disproportionately impaired incidental nor intentional location recognition memory relative to alcoholic controls. In contrast, mixed-etiology amnesics profited significantly from intentional location acquisition relative to incidental instructions, and were impaired somewhat in incidental, but not intentional, location memory relative to normal controls. We discuss these data in relation to Mayes' (1992) contextual memory deficit hypothesis and Hirst's (1982) automatic encoding deficit account, and propose an alternative framework in which the location memory deficit observed in mixed-etiology amnesics is interpreted as a disruption to the ability to bind item and location information.

Alcohol Amnestic Disorder↗

PubMed1996

Feature memory and binding in young and older adults.

Intact memory for complex events requires not only memory for particular features (e.g., item, location, color, size), but also intact cognitive processes for binding the features together. Binding provides the memorial experience that certain features belong together. The experiments presented here were designed to explicate these as potentially separable sources of age-associated changes in complex memory-namely, to investigate the possibility that age-related changes in memory for complex events arise from deficits in (1) memory for the kinds of information that comprise complex memories, (2) the processes necessary for binding this information into complex memories, or (3) both of these components. Young and older adults were presented with colored items located within an array. Relative to young adults, older adults had a specific and disproportionate deficit in recognition memory for location, but not for item or for color. Also, older adults consistently demonstrated poorer recognition memory for bound information, especially when all features were acquired intentionally. These feature and binding deficits separately contribute to what have been described as older adults' context and source memory impairments.

Adolescent↗

PubMed1995

Spectroscopic identification of the heme axial ligation of cytochrome b558 in the NADPH oxidase of porcine neutrophils.

The combination of electron paramagnetic resonance (EPR), near-infrared magnetic circular dichroism (NIR-MCD) and resonance Raman (RR) spectroscopies at cryogenic temperatures has been used to identify the axial heme ligation of the low spin cytochrome b558 component of NADPH oxidase from porcine blood neutrophils. The EPR and NIR-MCD results indicate the presence of two distinct forms in frozen solution; one with a low field g-value at 3.23 and porphyrin(pi)-to-Fe(III) charge transfer maximum at 1660 nm and the other a low field g-value at 3.00 and porphyrin(pi)-to-Fe(III) charge transfer maximum at 1510 nm. On the basis of these properties and the RR studies, both are attributed to forms of cytochrome b558 with bis-histidine axial ligation. The origin of the observed heterogeneity, the location and identity of the specific histidines involved in ligating the heme, and the role of the heme prosthetic group in O2- production are discussed in light of these results.

Animals↗

PubMed1995

The "nitrogenase-protective" FeSII protein of Azotobacter vinelandii: overexpression, characterization, and crystallization.

The Azotobacter vinelandii FeSII protein confers conformational protection to nitrogenase by binding to the MoFe and Fe proteins under periods of oxidative stress to create an inactive but O2-stabilized tripartite complex. In this work the FeSII protein has been overexpressed in Escherichia coli, and the recombinant protein has been purified to homogeneity, crystallized, and characterized in terms of its functional, spectroscopic, and redox properties. The recombinant protein is a homodimer and is expressed as a holoprotein with one [2Fe-2S]2+,+ cluster in each subunit. It is shown to be functional in reconstituting an O2-stable nitrogenase complex in vitro. Spectroscopic studies using the combination of UV-visible absorption, CD, and variable temperature MCD, EPR, and resonance Raman indicate that the [2Fe-2S]2+,+ cluster is coordinated exclusively by cysteine residues. The arrangement of coordinating cysteines in the primary sequence and the EPR properties of the [2Fe-2S]+ cluster (g = 2.04, 1.95, 1.88) are very similar to those of chloroplast ferredoxins. However, the variable-temperature MCD, resonance Raman, and redox properties (Em = -262 +/- 10 mV based on dye-mediated EPR redox titrations) are more characteristic of hydroxylase-type ferredoxins such as adrenodoxin. In contrast to chloroplast-type ferredoxins, the vibrational properties of the [2Fe-2S]2+,+ cluster in the FeSII protein indicate that none of the cysteinyl Fe-S-C-C dihedral angles are close to 180 degrees and that the cluster is not exposed to solvent. Preliminary X-ray diffraction analysis indicates that the protein crystallizes in an orthorhombic space group with unit cell dimensions a = 135 A, b = 135 A, and c = 38 A and that there are at least two dimers per asymmetric unit.

Azotobacter vinelandii↗

PubMed1995

Effect of cysteine to serine mutations on the properties of the [4Fe-4S] center in Escherichia coli fumarate reductase.

Site-directed mutants of Escherichia coli fumarate reductase in which FrdB Cys148, Cys151, Cys154, and Cys158 are replaced individually by Ser have been constructed and overexpressed in a strain of E. coli lacking a wild-type copy of fumarate reductase and succinate dehydrogenase. The consequences of these mutations on bacterial growth, enzymatic activity, and the EPR properties of the constituent iron-sulfur clusters have been investigated. The Cys154Ser and Cys158Ser FrdB mutations result in enzymes with negligible activity that have largely dissociated from the cytoplasmic membrane and consequently are incapable of supporting cell growth under conditions requiring a functional fumarate reductase. EPR studies indicate that these effects are associated with loss of both the [3Fe-4S] and [4Fe-4S] clusters. In contrast the Cys148Ser and Cys151Ser FrdB mutations result in functional membrane bound enzymes that are able to support growth under anaerobic and aerobic conditions. EPR studies of these mutants indicate that all three of the constituent Fe-S clusters are assembled, and the redox and spectroscopic properties of the [2Fe-2S] and [3Fe-4S] clusters are unchanged compared to the wild-type enzyme. In both mutants the [4Fe-4S] cluster is assembled with one non-cysteinyl ligand, and the available data suggest serinate coordination. The physicochemical consequences are perturbation of the intercluster spin interaction between the S = 1/2 [4Fe-4S]+ and S = 2 [3Fe-FS]0 clusters and a 60-mV decrease in redox potential for the [4Fe-FS]2+,+ cluster in the FrdB Cys148Ser mutant, and a S = 1/2 to S = 3/2 spin state conversion for the [4Fe-4S]+ cluster and a 72-mV decrease in redox potential for the [4Fe-4S]2+,+ cluster in the FrdB Cys151Ser mutant. Taken together with the previous FrdB Cys to Ser mutagenesis results [Werth, M. T., Cecchini, G., Manodori, A., Ackrell, B. A. C., Schröder, I., Gunsalus, R. P., & Johnson, M. K. (1990) Proc. Natl. Acad. Sci. U.S.A. 87, 8965-8969; Manodori, A., Cecchini, G., Schröder, I., Gunsalus, R. P., Werth, M. T., & Johnson, M. K. (1992) Biochemistry 31, 2703-2712], the results provide strong support for the proposal that all three clusters are located in the FrdB subunit with Cys57, Cys62, Cys65, and Cys77 ligating the [2Fe-2S] cluster, Cys148, Cys151, Cys154, and Cys214 ligating the [4Fe-4S] cluster, and Cys158, Cys204, and Cys210 ligating the [3Fe-4S] cluster. The role of the low potential [4Fe-4S] cluster in mediating electron transfer from menaquinol to the FAD active site is discussed in light of these mutagenesis results.

Amino Acid Sequence↗

PubMed1995

Spectroscopic identification of the axial ligands of cytochrome b560 in bovine heart succinate-ubiquinone reductase.

The axial ligands of low potential cytochrome b560 in the five subunit bovine heart succinate-ubiquinone reductase complex and in the isolated quinone binding proteins have been investigated using EPR and near-infrared magnetic circular dichroism spectroscopies. The results are consistent with bis-histidine ligation with near-perpendicular imidazole rings for cytochrome b560 in the four-subunit complex. The pronounced changes in EPR properties that accompany isolation of the cytochrome-b560 containing quinone binding proteins, are attributed to perturbation of the orientation of the imidazole rings of the heme bis-histidine ligands, rather than a change in axial ligation.

Animals↗

PubMed1995

Electron spin resonance studies on neutrophil cytochrome b558. Evidence that low-spin heme iron is essential for O2-. generating activity.

Cytochrome b558 purified from pig neutrophils was studied to characterize the spin state of the heme iron in relation to its O2-. generating activity. ESR spectra of cytochrome b558 either from resting or stimulated neutrophils showed a low-spin hemoprotein with g1,2,3 of 3.2, 2.1, and 1.3 (estimated). At physiological pH, the oxidized cytochrome b558 is in a purely low-spin state. On lowering or raising pH from 7, the spin state changes to high-spin. The ESR spectrum of high-spin cytochrome b558 was identical to that of methemoglobin, suggesting that the axial-ligand type in both hemoproteins may be the same, i.e. histidine is the fifth ligand. The ratio of the low-spin to high-spin heme in cytochrome b558 was evaluated by magnetic circular dichroism spectroscopy. The pH of cytochrome b558 was varied to form different ratios of the low-spin to high-spin states of the heme, and its O2-. generating activity was examined in cell-free systems. O2-. forming activity decreased concomitant with loss of the low-spin heme, which provides direct evidence that the low-spin state of cytochrome b558 is essential to generate O2-. and the heme retains the low-spin state through the redox cycle.

Animals↗

PubMed1995

Delayed neuropathy in pigs induced by isofenphos.

In 1990 an outbreak of ataxia occurred in over 700 pigs in the north of England. Epidemiological studies demonstrated that the disorder was associated with the consumption of feed from a particular supplier and that one component (wheat screenings) was common to the batch of feed with which the ataxia was associated. An analysis of the feed demonstrated the presence of an organophosphorus pesticide, later identified as isofenphos, a pesticide not approved for use in the United Kingdom. The wheat screenings had been imported from France and the warehouse in which they had been stored was contaminated with isofenphos, which is approved for restricted use in France. Isofenphos is known to cause delayed neuropathy. The dose to which the pigs were theoretically exposed would be expected to have resulted in neuropathy (manifested as ataxia).

Acetylcholinesterase↗

PubMed1995

Neuropathy target esterase (NTE) and organophosphorus-induced delayed polyneuropathy (OPIDP): recent advances.

The identification of neuropathy target esterase (NTE) as the site for initiation of organophosphorus-induced delayed polyneuropathy (OPIDP) has led to informative acute and chronic neurotoxicity tests (adopted by OECD and EPA), to structure/activity and in vitro/in vivo predictions, and to a sound basis for extrapolations to man. Purification of the sodium dodecyl sulphate (SDS)-denatured 155-kDa sub-unit of NTE has enabled partial sequencing and molecular biological studies. A MAb to the chicken brain sub-unit and PAbs to synthetic peptides have been raised: preliminary experiments suggest that one is effective for immunohistochemistry of frozen tissue. cDNA libraries are being screened with synthetic oligonucleotides, polymerase chain reaction (PCR)-developed primers, and with Ab in order to obtain cloned NTE. Previous studies of NTE in vivo have not revealed its normal physiological function or the route from inhibition to degeneration of axons, but the current progress in molecular biology of NTE is applicable to study of the function of normal and organophosphorus (OP)-modified NTE in cultured neural cells.

Animals↗

PubMed1995

Semantic relations and Alzheimer's disease: an early and disproportionate deficit in functional knowledge.

This experiment explored knowledge of four types of semantic relations (superordinate category, part, property, and function) in Alzheimer's disease (AD) subjects and age- and education-matched controls. Moderate AD subjects showed the greatest disruption on functional relations, intermediate disruption on part and property relations, and the least disruption on category relations; mild AD subjects showed a similar pattern but significant deficits only on functions. We suggest that the disproportionate deficit on functions reflects a greater cognitive complexity of functions than other semantic relations that renders them more vulnerable either to disrupted processing or to structural degradation of the network of associations among semantic concepts.

Aged↗

PubMed1995

Aging and single versus multiple cues in source monitoring.

Participants heard words said by 2 speakers and later decided who said each word. The authors varied the perceptual distinctiveness of the speakers and the distinctiveness of the cognitive operations participants performed on the words. Relative to younger adults, older adults had significantly lower source monitoring scores when perceptual or cognitive operations conditions were similar but not when either cue was more distinctive. Combining cues did not affect source monitoring of younger adults but hurt older adults' performance relative to the distinctive perceptual condition. Evidently, older adults generate cognitive cues at the expense of encoding perceptual cues; any deficit in binding perceptual and semantic information disadvantages them more in source monitoring than in old/new recognition. There was no correlation between neuropsychological tests assessing frontal function and source monitoring in older adults.

Adolescent↗

PubMed1995

Effects of elemental sulfur on the metabolism of the deep-sea hyperthermophilic archaeon Thermococcus strain ES-1: characterization of a sulfur-regulated, non-heme iron alcohol dehydrogenase.

The strictly anaerobic archaeon Thermococcus strain ES-1 was recently isolated from near a deep-sea hydrothermal vent. It grows at temperatures up to 91 degrees C by the fermentation of peptides and reduces elemental sulfur (S(o)) to H2S. It is shown here that the growth rates and cell yields of strain ES-1 are dependent upon the concentration of S(o) in the medium, and no growth was observed in the absence of S(o). The activities of various catabolic enzymes in cells grown under conditions of sufficient and limiting S(o) concentrations were investigated. These enzymes included alcohol dehydrogenase (ADH); formate benzyl viologen oxidoreductase; hydrogenase; glutamate dehydrogenase; alanine dehydrogenase; aldehyde ferredoxin (Fd) oxidoreductase; formaldehyde Fd oxidoreductase; and coenzyme A-dependent, Fd-linked oxidoreductases specific for pyruvate, indolepyruvate, 2-ketoglutarate, and 2-ketoisovalerate. Of these, changes were observed only with ADH, formate benzyl viologen oxidoreductase, and hydrogenase, the specific activities of which all dramatically increased in cells grown under S(o) limitation. This was accompanied by increased amounts of H2 and alcohol (ethanol and butanol) from cultures grown with limiting S(o). Such cells were used to purify ADH to electrophoretic homogeneity. ADH is a homotetramer with a subunit M(r) of 46,000 and contains 1 g-atom of Fe per subunit, which, as determined by electron paramagnetic resonance analyses, is present as a mixture of ferrous and ferric forms. No other metals or acid-labile sulfide was detected by colorimetric and elemental analyses. ADH utilized NADP(H) as a cofactor and preferentially catalyzed aldehyde reduction. It is proposed that, under So limitation, ADH reduces to alcohols the aldehydes that are generated by fermentation, thereby serving to dispose of excess reductant.

Alcohol Dehydrogenase↗

PubMed1995

Growth and virulence of a complement-activation-negative mutant of Streptococcus pneumoniae in the rabbit cornea.

Our previous work has demonstrated the importance of pneumolysin in the virulence of S. pneumoniae in a rabbit intracorneal model. This was accomplished by showing that deletion of the gene encoding pneumolysin resulted in reduced virulence, whereas restoration of the wild-type gene resulted in restoration of the virulent phenotype. To assess the importance of a particular domain in the pneumolysin molecule, we have now constructed a strain which produces a pneumolysin molecule which is hemolytic but which bears a site-specific mutation in the domain known to be associated with the complement-activating properties of this molecule. Comparison of the virulence of this strain with that of a strain bearing the wild-type gene showed statistically significantly lower total slit lamp examination (SLE) scores at 12, 18, 24, and 36 h (particularly with respect to fibrin formation), but no difference at 48 h. Determination of colony forming units (CFU) in eyes infected with the two strains showed approximately 10(6) bacteria per cornea until 36 h. Between 36 and 48 h, the bacteria were almost completely cleared with very few bacteria recoverable at the later time point. The loss of virulence observed with this mutation in the complement-activation domain of pneumolysin, though less than that observed with the gene deletion mutant, suggests that complement activation by pneumolysin has a significant role in the pathology observed in this model of corneal infection.

Amino Acid Sequence↗

PubMed1995

Comparison of three methods of measurement of pulmonary artery catheter readings in critically ill patients.

BACKGROUND: Pulmonary artery catheter readings are critical for clinical decision making and therapeutic intervention in critically ill patients. Research data of digital display versus graphic strip chart recording of hemodynamic pressures during spontaneous breathing and mechanical ventilation are inconclusive. OBJECTIVES: To compare three methods of measurement of hemodynamic pressure readings from the pulmonary artery catheter in critically ill patients during mechanical ventilation and spontaneous breathing. METHODS: A nonrandomized, repeated-measures design was used to compare hemodynamic pressures (right atrial, systolic, diastolic, and wedge pressures) from the pulmonary artery catheter in cardiovascular patients during mechanical ventilation (n = 25) and again during spontaneous breathing (n = 19). RESULTS: Using repeated measures analysis of variance, statistically significant differences were noted in the pulmonary artery diastolic, wedge, and right atrial pressure during mechanical ventilation. During spontaneous breathing, significant differences occurred in pulmonary artery systolic and wedge pressures only. No statistically significant difference occurred in the systolic pressure during mechanical ventilation, or the pulmonary artery diastolic and right atrial pressures during spontaneous breathing. CONCLUSIONS: The results of this study indicate that graphic recording is the most reliable means of measuring hemodynamic pressure at end-expiration. Further research is needed to validate these findings with other models of monitoring equipment and other patient populations.

Adult↗

PubMed1994

nifU gene product from Azotobacter vinelandii is a homodimer that contains two identical [2Fe-2S] clusters.

The nifU gene product is required for the full activation of the metalloenzyme nitrogenase, the catalytic component of biological nitrogen fixation. In the present work, a hybrid plasmid that contains the Azotobacter vinelandii nifU gene was constructed and used to hyperexpress the NIFU protein in Escherichia coli. Recombinant NIFU was purified to homogeneity and was found to be a homodimer of 33-kDa subunits with approximately two Fe atoms per subunit. The combination of UV/visible absorption, variable-temperature magnetic circular dichroism, EPR, and resonance Raman spectroscopies shows the presence of a [2Fe-2S]2+,+ center (Em = -254 mV) with complete cysteinyl coordination in each subunit. The electronic, magnetic, and vibrational properties of the [2Fe-2S]2+,+ center do not conform to those established for any of the spectroscopically distinct types of 2Fe ferredoxins. These distinctive properties appear to be a consequence of a novel arrangement of coordinating cysteinyl residues in NIFU, and the residues likely to be involved in cluster coordination are discussed in light of primary sequence comparisons to other putative [2Fe-2S] proteins. The observed physicochemical properties of NIFU and its constituent [2Fe-2S] cluster also provide insight into the role of this protein in nitrogenase metallocluster biosynthesis.

Azotobacter vinelandii↗

PubMed1994

Properties of the iron-sulfur center in the 25-kilodalton subunit of the proton-translocating NADH-quinone oxidoreductase of Paracoccus denitrificans.

The 25-kDa subunit of the proton-translocating NADH-quinone oxidoreductase (NDH-1) of Paracoccus denitrificans has been expressed in Escherichia coli and purified to homogeneity. EPR studies of the reduced recombinant protein indicated that the expressed subunit contains a single [2Fe-2S] cluster (Yano, T., Sled', V. D., Ohnishi, T., and Yagi, T. (1994) Biochemistry 33, 494-499). In this report, the electronic, magnetic, and vibrational properties of the [2Fe-2S]2+,+ center have been investigated by the combination of absorption, circular dichroism, variable-temperature magnetic circular dichroism, electron paramagnetic resonance, and resonance Raman spectroscopies and compared with a range of simple [2Fe-2S]-containing proteins. The results are consistent with coordination by two cysteinyl residues at both the reducible and nonreducible iron sites and reveal a striking similarity between the properties of the [2Fe-2S] cluster in the P. denitrificans NDH-1 25-kDa subunit and those of the subclass of ferredoxin-type [2Fe-2S] centers typified by Clostridium pasteurianum 2Fe ferredoxin. The four cyteines residues involved in cluster ligation in these proteins have been tentatively identified based on sequence homology considerations.

Amino Acid Sequence↗

PubMed1994

Synthesis and characterization of a biotinylated organophosphorus ester for detection and affinity purification of a brain serine esterase: neuropathy target esterase.

We have synthesized a novel stable precursor, saligenin phosphorotrichloridate, which, on reaction with N-monobiotinyldiamines, generates a series of biotinylated covalent inhibitors of serine esterases. A homologue designated S9B [1-(saligenin cyclic phospho)-9-biotinyldiaminononane] was selected to allow detection and rapid isolation of neuropathy target esterase (NTE). This enzyme is the primary target site for those organophosphorus esters (OPs) which cause delayed neuropathy. NTE comprises about 0.03% of the total protein in brain microsomal fractions and has resisted purification attempts over many years. S9B is a potent progressive inhibitor of NTE esteratic activity (second-order rate constant 1.4 x 10(7) M-1.min-1). Incubation of S9B with brain microsomes led to specific covalent labelling of NTE as determined by detection of a biotinylated 155 kDa polypeptide on Western blots. Specificity of S9B labelling was further demonstrated by inhibition with the neuropathic OP mipafox. Biotinyl-NTE in SDS-solubilized S9B-labelled microsomes was adsorbed on to avidin-Sepharose and subsequently eluted, yielding a fraction enriched approx. 1000-fold in NTE by a single step with recoveries of 30%. Essentially pure NTE was obtained after separation from two endogenous biotinylated polypeptides (120 and 70 kDa) in avidin-Sepharose eluates by preparative SDS/PAGE. Other biotinylated saligenin phosphoramidates derived from the same precursor may be useful for detection and isolation of other serine esterases and proteinases.

Animals↗

PubMed1994

Spectroscopic properties of desulfoferrodoxin from Desulfovibrio desulfuricans (ATCC 27774).

Desulfoferrodoxin, a non-heme iron protein, was purified previously from extracts of Desulfovibrio desulfuricans (ATCC 27774) (Moura, I., Tavares, P., Moura, J. J. G., Ravi, N., Huynh, B. H., Liu, M.-Y., and LeGall, J. (1990) J. Biol. Chem. 265, 21596-21602). The as-isolated protein displays a pink color (pink form) and contains two mononuclear iron sites in different oxidation states: a ferric site (center I) with a distorted tetrahedral sulfur coordination similar to that found in desulforedoxin from Desulfovibrio gigas and a ferrous site (center II) octahedrally coordinated with predominantly nitrogen/oxygen-containing ligands. A new form of desulfoferrodoxin which displays a gray color (gray form) has now been purified. Optical, electron paramagnetic resonance (EPR), and Mössbauer data of the gray desulfoferrodoxin indicate that both iron centers are in the high-spin ferric states. In addition to the EPR signals originating from center I at g = 7.7, 5.7, 4.1, and 1.8, the gray form of desulfoferrodoxin exhibits a signal at g = 4.3 and a shoulder at g = 9.6, indicating a high-spin ferric state with E/D approximately 1/3 for the oxidized center II. Redox titrations of the gray form of the protein monitored by optical spectroscopy indicate midpoint potentials of +4 +/- 10 and +240 +/- 10 mV for centers I and II, respectively. Mössbauer spectra of the gray form of the protein are consistent with the EPR finding that both centers are high-spin ferric and can be analyzed in terms of the EPR-determined spin Hamiltonian parameters. The Mössbauer parameters for both the ferric and ferrous forms of center II are indicative of a mononuclear high spin iron site with octahedral coordination and predominantly nitrogen/oxygen-containing ligands. Resonance Raman studies confirm the structural similarity of center I and the distorted tetrahedral FeS4 center in desulforedoxin and provide evidence for one or two cysteinyl-S ligands for center II. On the basis of the resonance Raman results, the 635 nm absorption band that is responsible for the gray color of the oxidized protein is assigned to a cysteinyl-S-->Fe(III) charge transfer transition localized on center II. The novel properties and possible function of center II are discussed in relation to those of mononuclear iron centers in other enzymes.

Desulfovibrio↗