Biomedical subjects

M K Johnson

Publications and source records attributed to M K Johnson.

At least 109 records · Page 6Linked to original sources

PubMed1994

Spectroscopic characterization of flavocytochrome c-552 from the photosynthetic purple sulfur bacterium Chromatium vinosum.

The identities of the axial ligands to the two hemes of the flavocytochrome c-552 isolated from the photosynthetic purple sulfur bacterium Chromatium vinosum have been investigated by visible/near-infrared absorption and magnetic circular dichroism (MCD) spectroscopies, with parallel electron paramagnetic resonance (EPR) studies. One of the hemes has histidine and methionine as axial ligands and has a local environment that is relatively insensitive to the composition of the bulk medium. The second heme, the local environment of which is sensitive to changes in the composition of the bulk medium, exists as a mixture of two forms, only one of which has histidine/methionine axial ligation. On the basis of its EPR characteristics, the other form most likely has histidine/lysine axial ligation. In aqueous solution near neutral pH, more than half of the second heme is present as the histidine/lysine form, while in 50:50 water/ethylene glycol the histidine/methionine form is the dominant one.

Chromatium↗

PubMed1994

Human ferrochelatase is an iron-sulfur protein.

Recombinant human ferrochelatase has been expressed in Escherichia coli and purified to homogeneity. Metal analyses revealed approximately 2 mol of non-heme Fe per mol of the purified enzyme (M(r) = 40,000). The UV-visible absorption spectrum of the purified enzyme consists of a protein absorption at 278 nm (epsilon approximately 90,000 M-1 cm-1) and bands at 330 nm (epsilon approximately 24,000 M-1 cm-1), 460 nm (shoulder, epsilon approximately 11,000 M-1 cm-1), and 550 nm (shoulder, epsilon approximately 9000 M-1 cm-1) that are indicative of a [2Fe-2S]2+ cluster. The spectra show an additional band at 415 nm that varied in intensity for different preparations and is attributed, at least in part, to a minor component of enzyme-associated high-spin Fe(III) heme. The presence of a single [2Fe-2S]2+,+ cluster as a redox active component of human ferrochelatase was confirmed by variable-temperature MCD and EPR studies of the dithionite-reduced enzyme which showed the presence of a S = 1/2 [2Fe-2S]+ cluster in addition to residual high spin Fe(II) heme. The reduced enzyme exhibits a S = 1/2 EPR signal, g = 2.00, 1.94, 1.91 accounting for 0.75 +/- 0.25 spins/molecule, that readily saturates at low microwave powers below 10 K but is observable without significant broadening at temperatures up to 100 K. The Fe-S cluster is labile and gradually disappears over period of 24 h, with concomitant loss of enzyme activity, when the enzyme is stored aerobically at 4 degrees C.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗

PubMed1994

Time-course studies of reality monitoring and recognition.

Two studies used a response-signal procedure to explore the time course of source-monitoring judgments about perceived and imagined events. Ss judged whether probe words corresponded to pictures that had previously been seen or imagined or were new. Old-new recognition accuracy grew to significant levels before reality-monitoring accuracy, supporting the notion that source monitoring requires more of or a different type of information than does old-new recognition. Also, source identification accuracy developed more quickly for imagined items than for perceived items. This difference in time-course functions is consistent with the idea that memories for perceived and imagined events differ in the relative amounts of various types of information they include (Johnson & Raye, 1981) and that these different types of information may revive or become available to source attribution mechanisms at different rates or may be differentially salient during reality monitoring.

Humans↗

PubMed1994

Aging and the effects of affective and factual focus on source monitoring and recall.

Pairs of young and elderly Ss participated in a short play and were then instructed to talk about factual or affective aspects of the play or to talk about the play without any particular focus suggested. In both the affective and control condition, older adults' ability to discriminate what they had said from what the other person had said was poorer than that of young adults. In contrast, when induced to focus on the factual content of the events, older Ss' source monitoring improved, and the age difference was reduced. The pattern was similar when Ss' ability to discriminate what they had said from what they had thought was examined. Furthermore, affective focus lowered the overall level of recall for both young and older Ss and led older Ss, in particular, to introduce more elaborations into their recall. The possibility that age difference in remembering content and source are related to type of focus is discussed.

Adolescent↗

PubMed1994

Mineral content of Louisiana white-tailed deer.

Water, lipid, and mineral composition were determined for metacarpals, phalanges, livers, and antlers from 219 free-ranging white-tailed deer (Odocoileus virginianus) collected on six properties in East Feliciana Parish, Louisiana (USA) to provide baseline data and to identify sources of composition variation. Metacarpal and phalangeal composition varied primarily with deer age; liver composition varied with deer sex.

Age Factors↗

PubMed1993

The role and properties of the iron-sulfur cluster in Escherichia coli dihydroxy-acid dehydratase.

Dihydroxy-acid dehydratase has been purified from Escherichia coli and characterized as a homodimer with a subunit molecular weight of 66,000. The combination of UV visible absorption, EPR, magnetic circular dichroism, and resonance Raman spectroscopies indicates that the native enzyme contains a [4Fe-4S]2+,+ cluster, in contrast to spinach dihydroxy-acid dehydratase which contains a [2Fe-2S]2+,+ cluster (Flint, D. H., and Emptage, M. H. (1988) J. Biol. Chem. 263, 3558-3564). In frozen solution, the reduced [4Fe-4S]+ cluster has a S = 3/2 ground state with minor contributions from forms with S = 1/2 and possibly S = 5/2 ground states. Resonance Raman studies of the [4Fe-4S]2+ cluster in E. coli dihydroxy-acid dehydratase indicate non-cysteinyl coordination of a specific iron, which suggests that it is likely to be directly involved in catalysis as is the case with aconitase (Emptage, M. H., Kent, T. A., Kennedy, M. C., Beinert, H., and Münck, E. (1983) Proc. Natl. Acad. Sci. U.S.A. 80, 4674-4678). Dihydroxy-acid dehydratase from E. coli is inactivated by O2 in vitro and in vivo as a result of oxidative degradation of the [4Fe-4S]cluster. Compared to aconitase, the oxidized cluster of E. coli dihydroxy-acid dehydratase appears to be less stable as either a cubic or linear [3Fe-4S] cluster or a [2Fe-2S] cluster. Oxidative degradation appears to lead to a complete breakdown of the Fe-S cluster, and the resulting protein cannot be reactivated with Fe2+ and thiol reducing agents.

Amino Acid Sequence↗

PubMed1993

Variable temperature magnetic circular dichroism studies of reduced nitrogenase iron proteins and [4Fe-4S]+ synthetic analog clusters.

Variable temperature magnetic circular dichroism (VTMCD) and EPR spectroscopies have been used to investigate the ground and excited-state properties of [4Fe-4S]+ clusters in Mo- and V-nitrogenase Fe-proteins from Azotobacter vinelandii and two synthetic analog clusters, [Fe4S4(SEt)4]3- and [Fe4S4(SC6H11)4]3-. The results indicate similar [4Fe-4S]+ clusters with analogous S = 1/2 and S = 3/2 ground states in both Fe-proteins. However, the Fe-proteins do differ in terms of the medium effects on the S = 1/2 and S = 3/2 spin mixtures in frozen solution. By utilizing medium effects in both Fe-proteins, the VTMCD characteristics of both the S = 1/2 and S = 3/2 forms of the [4Fe-4S]+ have been determined. Together with the VTMCD studies of [Fe4S4(SEt)4]3- and [Fe4S4(SC6H11)4]3-, which are shown to be predominantly S = 1/2 and 3/2, respectively, in frozen DMF/toluene solutions, the results demonstrate that the form of the VTMCD spectra provides a means of identifying and distinguishing S = 1/2 and S = 3/2 [4Fe-4S]+ clusters. Ground state zero-field splitting parameters for the S = 3/2 clusters are determined for both Fe-proteins. In addition to spin state heterogeneity, samples of the Mo-nitrogenase Fe-protein in the presence of 50% (v/v) ethylene glycol were found to exhibit heterogeneity in the S = 1/2 resonance. A rapidly relaxing axial resonance, g perpendicular = 1.94 and g parallel = 1.82, was observed in addition to the characteristic rhombic resonance, g = 2.05, 1.94 and 1.87. The origin of the heterogeneity exhibited by [4Fe-4S]+ clusters in frozen solution is discussed in light of these results.

Circular Dichroism↗

PubMed1993

Interactions in vitro of some organophosphoramidates with neuropathy target esterase and acetylcholinesterase of hen brain.

For organophosphates or phosphonates to initiate delayed neuropathy two steps are necessary: (1) progressive covalent reaction with neuropathy target esterase (NTE) to produce a form of inhibited NTE which can be reactivated by incubation with aqueous potassium fluoride (KF) and (2) progressive "aging" of inhibited NTE to a form which can no longer be reactivated by KF. However, it has been shown recently that certain N-unsubstituted organophosphoro-monoamidates (analogues of methamidophos) cause delayed neuropathy even though the inhibited NTE appeared not to have aged (Johnson et al. (1991). Arch. Toxicol., 65, 618-624). In order to study the generality of this phenomenon, we have examined some N-substituted compounds. We report in vitro studies of inhibition and reactivation and aging of both NTE and acetylcholinesterase (AChE) prior to toxicological tests. All the compounds studied were less inhibitory to both NTE and AChE in concentrated rather than in dilute suspensions of EDTA-washed brain particles without added cofactors. There was an apparent disposal of up to 100 mumoles of test compound by particles from 95 mg hen brain, which is far greater than can be explained by covalent binding. The activity is distinct from calcium-dependent "A" esterase. Several N-alkyl phosphoromonoamidates were found to be potent and selective inhibitors of NTE: second-order rate constant for O-n-pentyl N-benzylphosphoramido-fluoridate (Cmpd 6) = 5.6 x 10(7) M-1 min-1 at 37 degrees, which is about 100x higher than for acetylcholinesterase (AChE). Inhibited NTE and AChE from several chiral phosphoromono-amidates did not reactivate spontaneously (21 hours at 37 degrees). Virtually 100% reactivation by KF of AChE inhibited by phosphoromonoamidates was achieved at all times tested. Acetylcholinesterase inhibited by 2,5-dichlorophenyl N,N'-di-n-butylphosphorodiamidate was 42-56% reactivated by incubation with KF (192 mM in pH 5.2 buffer for 30 minutes at 37 degrees). We believe this is the first report of reactivation of any enzyme after inhibition by a phosphorodiamidate. For NTE inhibited by tabun (O-ethyl N-dimethylphosphoroamidocyanidate), virtually complete and rapid aging (t1/2 = 5.5-8.4 minutes) was observed. Consistent but only partial reactivation by KF was achieved 2 or more hours after inhibition of NTE by Cmpd 6 or by its 2,6-difluoro-analogue (Cmpd 7). However, a small but significant aging (approximately 15-20% loss of reactivatability) was measured soon after a 1 minute inhibition by Cmpd 7, but no further change occurred in 21 hours.(ABSTRACT TRUNCATED AT 400 WORDS)

Acetylcholinesterase↗

PubMed1993

Purification and characterization of Klebsiella aerogenes UreE protein: a nickel-binding protein that functions in urease metallocenter assembly.

The Klebsiella aerogenes ureE gene product was previously shown to facilitate assembly of the urease metallocenter (Lee, M.H., et al., 1992, J. Bacteriol. 174, 4324-4330). UreE protein has now been purified and characterized. Although it behaves as a soluble protein, UreE is predicted to possess an amphipathic beta-strand and exhibits unusually tight binding to phenyl-Sepharose resin. Immunogold electron microscopic studies confirm that UreE is a cytoplasmic protein. Each dimeric UreE molecule (M(r) = 35,000) binds 6.05 + 0.25 nickel ions (Kd of 9.6 +/- 1.3 microM) with high specificity according to equilibrium dialysis measurements. The nickel site in UreE was probed by X-ray absorption and variable-temperature magnetic circular dichroism spectroscopies. The data are most consistent with the presence of Ni(II) in pseudo-octahedral geometry with 3-5 histidyl imidazole ligands. The remaining ligands are nitrogen or oxygen donors. UreE apoprotein has been crystallized and analyzed by X-ray diffraction methods. Addition of nickel ion to apoprotein crystals leads to the development of fractures, consistent with a conformational change upon binding nickel ion. We hypothesize that UreE binds intracellular nickel ion and functions as a nickel donor during metallocenter assembly into the urease apoprotein.

Amino Acid Sequence↗

PubMed1993

Hydrolysis of some organophosphorus dichlorophenyl esters by hen brain homogenates and rabbit serum compared with hydrolysis of paraoxon.

The hydrolysis of four organophosphorus dichlorophenyl esters and of paraoxon was studied in hen brain homogenates and in rabbit serum. All compounds were hydrolysed by both preparations, but the rates were different in the two preparations. EDTA inhibited the hydrolysis almost completely in rabbit serum, but had only a small effect on the hydrolysis in hen brain homogenates.

Animals↗

PubMed1993

Stereo-specific degradation of the R-(+) isomer of O-n-hexyl-S-methylphosphorothioamidate catalysed by rabbit serum.

Resolved isomers of O-n-hexyl-S-methylphosphorothioamidate (HXM) which had been synthesised by separate stereospecific routes were analysed by chiral glc: about 2-3% of R-(+) isomer was found in the S-(-) sample and accounted for nearly all the inhibitory power against neuropathy target esterase. Incubation of racemic HXM with rabbit serum led to slow but very specific disposal of R-(+) isomer to undetectable levels with very slight loss of S-(-): the rate of disposal was roughly estimated to be about 1% of the published rate of hydrolysis of paraoxon. Incubation with crystalline chymotrypsin caused a preferential but not totally selective disposal of S-(-) isomer.

Animals↗

PubMed1993

Symposium introduction: retrospect and prospects for neuropathy target esterase (NTE) and the delayed polyneuropathy (OPIDP) induced by some organophosphorus esters.

This article introduces a Symposium devoted to Neuropathy Target Esterase (NTE). The characteristics of the disorder known as OPIDP are described and the steps by which NTE was identified as the target are summarised. Studies with many organophosphates, phosphinates and chiral phosphonates are entirely consistent with a 2-step process of initiation referred to as 'NTE (70-80%) aging': about 70-80% of available nervous system NTE is first covalently phosphylated causing inhibition of esterase activity, and then the molecules of inhibited NTE undergo a covalent bond-cleavage leaving a negative charge in the region of the still-bound phosphorus. This understanding has clarified structure/activity studies of neuropathic potential of OP esters and is now routinely applied in toxicological evaluations for regulatory purposes. However, the biological function of NTE has remained a mystery. Prospective views of the role of NTE are presented by different authors. Attempts to isolate catalytically active or radiolabelled inhibited NTE are near to success. Since the Symposium, complete isolation of NTE affinity-purified from hen brain has been reported (see M.K. Johnson & P. Glynn, Toxicologist, 13 (1993) 211, Abstr. 773). Some minor, but possibly significant, differences in properties of a soluble and a membrane-bound form of NTE in sciatic nerve has been identified. The nature of the disturbance brought about by covalent binding of organophosphoramidates at the active site of NTE and the discovery that 'non-aging' inhibitors of NTE can promote neuropathy in hens given a sub-neuropathic dose of neuropathic OPs has led to a concept of NTE inhibitors having a range of 'partial agonist' effects at the covalent binding site. Evidence is emerging that the promotion target may be 'cousin-of NTE' with very similar inhibition characteristics and a function in the processes of response to or repair of axonal damage.

Animals↗

PubMed1993

Molecular characterisation of neuropathy target esterase: proteolysis of the [3H]DFP-labelled polypeptide.

Neuropathy target esterase (NTE) in hen brain membranes can be labelled with tritiated di-isopropylfluorophosphate ([3H]DFP) and appears to be associated with a 155-kDa polypeptide. Using preparative SDS-PAGE, we have obtained preparations in which [3H]DFP-labelled NTE comprises 2% of the total protein. Further purification of the 155-kDa polypeptide has proved difficult. We therefore attempted to use proteases to excise smaller [3H]DFP-labelled fragments which might be more amenable to fractionation. V8 protease treatment generated a labelled fragment of about 16 kDa which could be fractionated on SDS-PAGE and contained tritium attached to both site X (putatively the active site serine) and site Z (the residue to which an isopropyl moiety is transferred during aging of [3H]DFP-inhibited NTE). Papain and thermolysin treatments generated a small labelled peptide (< 10 kDa) which could be fractionated on reverse-phase HPLC and in which tritium was attached to site X but not site Z. N-terminal sequencing of the thermolysin-generated peptide fraction indicated sample heterogeneity but also suggested that the active site of NTE may contain the serine esterase consensus sequence: Gly-Glu-Ser-Xxx-Gly.

Amino Acid Sequence↗

PubMed1993

Reactivation of phosphorodiamidated acetylcholinesterase and neuropathy target esterase by treatment of inhibited enzyme with potassium fluoride.

It has been thought that the phosphorus-enzyme bond in inhibited esterases inhibited by such agents as mipafox (N,N'-di-iso-propylphosphorodiamidate) was refractory to reactivating agents either because an 'aging' reaction occurs soon after inhibition or because the bond was intrinsically very strong. We have found that both acetylcholinesterase (AChE) and neuropathy target esterase (NTE) which had been inhibited with either mipafox or with a di-n-butylphosphorodiamidate could be reactivated by prolonged treatment with aqueous potassium fluoride (KF): the reaction proceeded with first-order kinetics. Furthermore there was no time-dependent loss of reactivatability (aging). Di-isopropylphosphoro-butyrylcholinesterase could be fully reactivated by this treatment but after 18 h to allow aging the monoisopropyl phosphoro-enzyme was totally refractory to KF. We conclude that it is likely that the mipafox-enzyme bond in inhibited NTE and AChE is relatively strong but that aging has not occurred. The local disturbance around the active site of NTE caused by attachment of the phosphorodiamidate molecule appears to be sufficient to initiate delayed neuropathy without necessity for an 'aging' reaction.

Acetylcholinesterase↗

PubMed1993

Screening of O-ethyl O-4-nitrophenyl phosphoramidate (ENPP) for delayed neuropathic potential.

O-Ethyl-O-4-nitrophenylphosphoramidate is a short-acting anticholinesterase and a possible candidate for a prophylactic agent against nerve agents since human acetylcholinesterase inhibited by this agent undergoes rapid spontaneous reactivation which can be accelerated further, if necessary, by treatment with oximes. Doses of the agent > 1 mg/kg (s.c.) given to unprotected rats were fatal in a short time but 2 rats and one hen given 0.5 mg/kg survived. Hens given 2.5 or 4 mg/kg s.c. 20 min after prophylactic physostigmine + atropine survived acute effects and were killed 4.5 or 24 h later. Brain and spinal cord neuropathy target esterase levels of these hens were depressed only 4-10% compared with levels in brains from hens given only oxime + atropine or of undosed animals. Clinical signs of neuropathy were not seen in surviving birds observed for 3 weeks. It appears there would be negligible delayed neuropathic hazard associated with administration of O-ethyl-O-4-nitrophenylphosphoramidate at subacute doses.

Animals↗

PubMed1993

The R-(+)isomer of O-n-hexyl S-methyl phosphorothioamidate causes delayed neuropathy in hens after generation of a form of inhibited neuropathy target esterase (NTE) which can be reactivated ex vivo.

To initiate delayed neuropathy (DN) in adult hens organophosphates and phosphonates must inhibit most neural NTE and the inhibited NTE must undergo an 'aging' reaction. Phosphinates and those chiral isomers of phosphonates which produce non-aging NTE do not cause DN but act as prophylactic agents. Some racemic phosphoramidates cause DN although the inhibited NTE in autopsy samples can be reactivated in vitro (Johnson, Read and Vilanova, 1991, Arch. Toxicol., 65, 618-624). We now report that pure R(+)isomer of O-n-hexyl S-methyl phosphorothioamidate (5-20 mg/kg per os) caused slight acute effects but typical DN associated with high inhibition of NTE in brain, spinal cord and sciatic nerve (maximum by 6-24 h): the inhibited NTE was easily reactivated by KF (presumed not aged). For each dose the average residual NTE activity in the three tissues 24 h after dosing and the clinical ataxia severity on peak days 15-17 (score out of 4) was: 5 mg/kg: 13, 14, 27% (2,2,2,1); 10 mg/kg: 10, 14, 12%, (4,3,2); 15 mg/kg: 10,11,17%, (3,3,4); 20 mg/kg: 6, 10, 8% (3,3,3,2). The ability of this isomer and of other racemic phosphoramidates to initiate DN by covalent reaction at the active site of NTE (inhibition) without subsequent aging suggests that the chemistry (? charge distribution) in the region of the phosphorus atom determines that disturbance in the molecular environment of NTE which initiates DN.

Animals↗

PubMed1993

Prophylaxis against and promotion of organophosphate-induced delayed neuropathy by phenyl di-n-pentylphosphinate.

Phenyl di-n-pentylphosphinate (PPP) is a potent inhibitor of neuropathy target esterase (NTE) with negligible effect on acetylcholinesterase: I50S at 37 degrees C for 20 min and pH 8, respectively are 0.2 microM and > 2mM. PPP is not neuropathic. This is compatible with the fact that inhibited NTE is autopsy material from hens dosed with PPP can always be reactivated in vitro, presumably because no 'aging' reaction has occurred. PPP (10 mg/kg s.c.) given to hens up to 4 days before severely neuropathic doses (1.7 mg/kg) of diisopropylphosphorofluoridate (DFP) prevented neuropathic but not cholinergic effects of DFP. Hens given PPP 3 days after a sub-neuropathic dose of DFP (0.4 mg/kg) developed severe clinical neuropathy (clinical scores of 7 and 5 compared with DFP-plus-solvent scores 0,1,3). These prophylactic and promoting effects are similar to those exerted by phenylmethanesulphonyl fluoride (PMSF) at doses which inhibit NTE. In 3 out of 4 birds a pre-dose with PMSF (15 mg/kg) prevented the promoting effect of 120 mg/kg PMSF given after DFP.

Animals↗

PubMed1993

Source monitoring.

A framework for understanding source monitoring and relevant empirical evidence is described, and several related phenomena are discussed: old-new recognition, indirect tests, eyewitness testimony, misattributed familiarity, cryptomnesia, and incorporation of fiction into fact. Disruptions in source monitoring (e.g., from confabulation, amnesia, and aging) and the brain regions that are involved are also considered, and source monitoring within a general memory architecture is discussed. It is argued that source monitoring is based on qualities of experience resulting from combinations of perceptual and reflective processes, usually requires relatively differentiated phenomenal experience, and involves attributions varying in deliberateness. These judgments evaluate information according to flexible criteria and are subject to error and disruption. Furthermore, diencephalic and temporal regions may play different roles in source monitoring than do frontal regions of the brain.

Attention↗