Neurotoxicity of a spent phosphoric acid catalyst.
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Animals↗
Biomedical subjects
M K Johnson
Publications and source records attributed to M K Johnson.
The inhibition site of MPP+, the neurotoxic bioactivation product of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine is near the Q-binding site of NADH dehydrogenase.
The inhibition of NADH dehydrogenase by 1-methyl-4-phenylpyridinium (MPP+) leading to ATP depletion has been proposed to explain cell death in the expression of the neurotoxicity of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP). Electron paramagnetic resonance studies show no effect of MPP+ on the reduction of the iron-sulfur clusters of NADH dehydrogenase. Mitochondria inhibited by MPP+ were sonicated and both the NADH oxidase and the NADH-Q reductase activities were measured. NADH oxidase activity was not fully restored to control levels, but NADH-Q reductase activity was the same as that of the control. Neither succinate-oxidase nor succinate-Q reductase activities were inhibited. These data indicate that MPP+ interaction with NADH dehydrogenase interferes with the passage of electrons from the iron-sulfur cluster of highest potential to endogenous Q10 but that the inhibition can be relieved by the addition of a small, water-soluble Q analog. Inhibition at this site is sufficient to explain the inhibition of respiration and no inhibition of other mitochondrial functions was observed.
1-Methyl-4-phenyl-1,2,3,6-tetrahydropyridine↗
The influence of chirality on the delayed neuropathic potential of some organophosphorus esters: neuropathic and prophylactic effects of stereoisomeric esters of ethyl phenylphosphonic acid (EPN oxon and EPN) correlate with quantities of aged and unaged neuropathy target esterase in vivo.
Organophosphate-induced delayed polyneuropathy (OPIDP) is thought to result from organophosphorylation of neuropathy target esterase (NTE), followed by an "aging" of the phosphorylated NTE. Prophylactic against OPIDP should thus be achieved by production of an inhibited but "nonaging" NTE. Resolved stereoisomers of ethyl phenylphosphonic acid esters produce two forms of inhibited NTE; in vitro one form ages rapidly and the other only negligibly. The present study examined the in vivo effects of two preparations of incompletely resolved isomers of EPN oxon (ethyl 4-nitrophenyl phenylphosphonate) and its thionate on adult hen brain and spinal cord NTE and the relationship of inhibition and aging to the development of OPIDP. Single doses of the L-(-)-isomers (Preparation A, 7:3 proportion of isomers, or Preparation B, 9:1) caused severe neuropathy after doses which produced 70% aged inhibited NTE and mild effects after 50-60%. Single doses of the D-(+)-isomers produced either equal amounts of aged and unaged inhibited NTE (Preparation A) or predominantly unaged (Preparation B): the amount of aged was never more than 50% and no clinical OPIDP occurred. Doses of D-(+) which produced 50% unaged inhibited NTE were protective: challenge with the highly neuropathic phenyl saligenin cyclic phosphate did not cause OPIDP. All effects are consistent with the two-stage initiation process which requires both inhibition of NTE and subsequent modification of the protein by an "aging" process. Previously reported neuropathic effects of D-(+)-EPN probably reflect a substantial proportion of L-(-)-isomer present in the test material. Neuropathic studies with chiral OP esters should consider the possibility of production of protective unaged inhibited NTE in test animals.
Animals↗
Spectroscopic properties of siroheme extracted from sulfite reductases.
Siroheme has been extracted from sulfite reductases and its properties in aqueous solution have been investigated by optical absorption, electron paramagnetic resonance (EPR), and magnetic circular dichroism (MDC) spectroscopy. The absorption spectrum of siroheme exhibits a marked pH dependence, and two pK values, 4.2 and 9.0, were determined by pH titration in the range 2-12. The first pK (4.2) is thought to correspond to the ionization of the carboxylic acid side-chains on the tetrapyrrole rings, and the second pK (9.0) is attributed to displacement of the axial ligand chloride by hydroxide. The binding of the strong field ligands, CO, NO, and cyanide, were investigated by UV-visible absorption and, in the case of the cyanide complex, by low-temperature EPR and MCD spectroscopies. CO and NO were able to reduce and bind to siroheme without additional reducing agent. The EPR spectrum of the isolated siroheme (chloride-ferrisiroheme) exhibits an axial signal with g perpendicular = 6.0 and g parallel = 2.0, typical of high-spin ferric hemes (S = 5/2), whereas the cyanide-complexed siroheme exhibits an approximately axial signal with g perpendicular = 2.38 and g parallel = 1.76 that is indicative of a low-spin ferric heme (S = 1/2). The low-temperature MCD spectra and magnetization data for the as-isolated and cyanide-complexed ferrisiroheme are entirely consistent with the interpretation of the EPR spectra. The results for ferrosiroheme indicate that the siroheme remains high spin (S = 2) and low spin (S = 0) on reduction of the as-isolated and cyanide-complexed siroheme, respectively. The isolated siroheme expressed sulfite reductase activity but the assessable catalytic cycle was much less than that of the native enzyme, showing the importance of the protein environment.
Desulfovibrio↗
Is event frequency encoded automatically? The case of alcohol intoxication.
The four experiments reported show that sensitivity to event frequency is diminished by alcohol intoxication. In two experiments on confusion between presented and generated words, the function relating estimated to actual frequency was steeper for sober than intoxicated subjects. In two experiments on word-frequency estimates after a word-pronunciation task, the influence of alcohol intoxication was identified at the input as opposed to the test stage. We conclude that these findings are inconsistent with the idea that frequency-of-occurrence information is automatically encoded.
Adult↗
Molecular cloning, characterization, and complete nucleotide sequence of the gene for pneumolysin, the sulfhydryl-activated toxin of Streptococcus pneumoniae.
A recombinant lambda bacteriophage has been isolated that carries DNA from Streptococcus pneumoniae and expresses a potent hemolysin that has been shown to be pneumolysin, the sulfhydryl-activated toxin of the pneumococcus. Hemolytic activity is inhibited by cholesterol and neutralized by serum against streptolysin O. The cloned gene expresses two polypeptides (Mrs, 56,000 and 53,000) in an Escherichia coli in vitro transcription-translation system, and both are precipitated by the addition of anti-alveolysin serum and anti-streptolysin O serum in the presence of Staphylococcus aureus cells. Expression of pneumolysin occurs when the gene is cloned in both possible orientations in pUC8. The DNA sequence of a 5-kilobase ClaI fragment that carries the pneumolysin gene has been determined. An open reading frame was identified that encodes a polypeptide of 471 amino acids that is hydrophobic in character and has an N-terminal amino acid sequence which is identical to that deduced from amino acid sequencing of the purified protein. The predicted amino acid sequence of the polypeptide reveals a single cysteine residue located 44 residues from the C terminus. Putative promoter and ribosome binding sites have been identified 5' to the pneumolysin coding sequence.
Amino Acid Sequence↗
A benefit from mechanistic research in toxicology.
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Carboxylic Ester Hydrolases↗
Human learning and memory.
There have been several notable recent trends in the area of learning and memory. Problems with the episodic/semantic distinction have become more apparent, and new efforts have been made (exemplar models, distributed-memory models) to represent general knowledge without assuming a separate semantic system. Less emphasis is being placed on stable, prestored prototypes and more emphasis on a flexible memory system that provides the basis for a multitude of categories or frames of reference, derived on the spot as tasks demand. There is increasing acceptance of the idea that mental models are constructed and stored in memory in addition to, rather than instead of, memorial representations that are more closely tied to perceptions. This gives rise to questions concerning the conditions that permit inferences to be drawn and mental models to be constructed, and to questions concerning the similarities and differences in the nature of the representations in memory of perceived and generated information and in their functions. There has also been a swing from interest in deliberate strategies to interest in automatic, unconscious (even mechanistic!) processes, reflecting an appreciation that certain situations (e.g. recognition, frequency judgements, savings in indirect tasks, aspects of skill acquisition, etc) seem not to depend much on the products of strategic, effortful or reflective processes. There is a lively interest in relations among memory measures and attempts to characterize memory representations and/or processes that could give rise to dissociations among measures. Whether the pattern of results reflects the operation of functional subsystems of memory and, if so, what the "modules" are is far from clear. This issue has been fueled by work with amnesics and has contributed to a revival of interaction between researchers studying learning and memory in humans and those studying learning and memory in animals. Thus, neuroscience rivals computer science as a source of interdisciplinary stimulation. Research on topics such as memory for spatial location, the relation between memory and affect, and autobiographical memory reminds us that general theories of memory based on studies of verbal materials alone are limited. Investigating how people remember complex natural events should provide us with a larger set of memory phenomena to explain and consequently insight into a wider range of memory principles or a deeper understanding of the ones we already accept (e.g. the role of repetition, encoding specificity), including their functional significance for human behavior.(ABSTRACT TRUNCATED AT 400 WORDS)
Affect↗
Spectroscopic characterization of the number and type of iron-sulfur clusters in NADH:ubiquinone oxidoreductase.
The number and type of iron-sulfur clusters present in the NADH dehydrogenase of the mammalian respiratory chain were studied by a combination of low temperature magnetic circular dichroism (MCD) and quantitative electron paramagnetic resonance spectroscopies. MCD was used with the high molecular weight, soluble enzyme, and EPR was used with both the purified enzyme and Complex I (NADH:ubiquinone oxidoreductase). The results of the EPR experiments of the two types of preparations agreed with each other, as well as with the data in the literature for various types of membrane-bound preparations. The two methods gave concordant results showing the presence of one binuclear and of three tetranuclear NADH-reducible iron-sulfur clusters. Earlier studies using the cluster extrusion technique indicated a higher ratio of binuclear to tetranuclear clusters which may be explained by cluster interconversion during the extrusion process.
Animals↗
On the novel H2-activating iron-sulfur center of the "Fe-only" hydrogenases.
The two hydrogenases (I and II) of the anaerobic N2-fixing bacterium Clostridium pasteurianum (Cp) and the hydrogenases of the anaerobes Megasphaera elsdenii (Me) and Desulfovibrio vulgaris (strain Hildenborough, Dv), contain iron-sulfur clusters but not nickel. They are the most active hydrogenases known. All four enzymes in their reduced states give rise to EPR signals typical of [4Fe-4S]1+ clusters but exhibit novel EPR signals in their oxidized states. For example, Cp hydrogenase I exhibits a sharp rhombic EPR signal when oxidized under mild conditions but the enzyme is inactivated by over-oxidation and then exhibits an axial EPR signal. A similar axial signal is observed from mildly oxidized hydrogenase I after treatment with CO. EPR, Mössbauer and ENDOR spectroscopy indicate that the EPR signals from the oxidized enzyme and its CO derivative arise from a novel spin-coupled Fe center. Low temperature magnetic circular dichroism (MCD) studies reveal that an EPR-silent Fe-S cluster with S greater than 1/2 is also present in oxidized hydrogenase I. From a study of all spectroscopic properties of Cp, Dv, and Me hydrogenases, it is concluded that the H2-activating site of all four is a novel Fe-S cluster with S greater than 0 and integer, which in the oxidized state is exchange-coupled to a S = 1/2 species. The data are most consistent with the S = 1/2 species being a low spin Fe(III) center. The H2-activating site is susceptible to oxidative rearrangements to yield both active and inactive states of the enzyme. We discuss the possible implications of these finding to methods of enzyme oxidation and purification procedures currently used for hydrogenases.
Anaerobiosis↗
Recognition and recall in amnesics.
Although there is considerable agreement that performance in direct memory tasks (e.g., recall, recognition) is more disrupted by amnesia than performance in indirect memory tasks (e.g., mirror reading, word completion), one may be able to further circumscribe the deficit within the domain of direct memory tasks. The present article explores whether recall is disproportionately disrupted by amnesia compared to recognition. If amnesia affects memory uniformly across different direct memory measures, recall of normal controls should not differ from the recall of amnesics when recognition scores of these two groups are equated. On the other hand, if recall is disproportionately disrupted, normal recall should be superior to amnesic recall even when recognition is equated. The present study equated amnesic recognition with that of controls by providing amnesics with 8 s of study time and normal subjects with 0.5 s. Amnesics with Korsakoff's syndrome, amnesics with other etiologies, and appropriate controls were examined. Normal recall was superior to amnesic recall even when no differences were found in recognition. The results further specify the selective nature of amnesia.
Alcohol Amnestic Disorder↗
The high potential iron-sulfur center in Escherichia coli fumarate reductase is a three-iron cluster.
The fumarate reductase complex and soluble enzyme from Escherichia coli have been investigated by low temperature magnetic circular dichroism and electron paramagnetic resonance spectroscopies. The results confirm the presence of one [2Fe-2S] cluster and show that the high potential iron-sulfur center is a 3Fe cluster of the type found in bacterial ferredoxins. Since the 3Fe cluster is present in catalytically competent enzyme and does not appear to be involved in any type of cluster conversion under reducing conditions, we conclude that it is an intrinsic component of the functional enzyme. The significance of the results is discussed in relation to the published amino acid sequence and the iron-sulfur cluster composition of bacterial fumarate reductases.
Amino Acid Sequence↗
The prosthetic groups of succinate dehydrogenase: 30 years from discovery to identification.
Recent studies using magnetic circular dichroism at cryogenic temperatures, electron paramagnetic resonance (EPR) and linear electric field effect-EPR (LEFE) of succinate dehydrogenase in membranes and in soluble, homogeneous preparations demonstrated the presence of 3 different Fe-S clusters in the mammalian enzyme, as well as in a similar bacterial enzyme, fumarate reductase from Escherichia coli. There is one each of the 2Fe, 3Fe, and 4Fe clusters. Thus, succinate dehydrogenase is the first enzyme which has been shown to contain all 3 of these Fe-S clusters. The enzyme also contains 1 mol 8 alpha-[N(3)-histidyl]-FAD. It has taken the combined expertise of many laboratories and 15 years of effort to identify the flavin component, and nearly 3 decades to identify the Fe-S clusters. The data from physical methods appear to be internally consistent, in harmony with the results of chemical analysis, and provide a rational explanation for earlier results by the cluster extrusion method. There remains, however, a number of interesting and substantive questions for future investigations. This review traces the tortuous path, the many pitfalls and false leads, which have led us from the discovery of nonheme iron and 'bound' flavin in the enzyme to elucidation of their structures.
Chemistry↗
In vivo detection of a three iron cluster in fumarate reductase from Escherichia coli.
Escherichia coli with plasmid amplified expression of fumarate reductase was grown anaerobically on a medium containing fumarate and glycerol and investigated by electron paramagnetic resonance spectroscopy. Anaerobically harvested cells exhibit an EPR signal characteristic of a reduced [2Fe-2S] cluster. Anaerobic addition of fumarate results in diminution of the reduced [2Fe-2S] signal and the appearance of the EPR signal associated with the oxidized 3Fe cluster. The results provide the first evidence for a trinuclear iron-sulfur cluster that exists in vivo, and suggest that the 3Fe cluster in purified fumarate reductase samples is not an artifact of the isolation procedure. The significance of this observation is discussed in relation to the physiological relevance of trinuclear iron-sulfur clusters.
Circular Dichroism↗
Detection of a tetranuclear iron-sulfur center in fumarate reductase from Escherichia coli by electron paramagnetic resonance spectroscopy.
Soluble fumarate reductase and fumarate reductase complex from Escherichia coli have been investigated by electron paramagnetic resonance spectroscopy. Both succinate- and dithionite-reduced samples show signals associated with a [2Fe-2S]1+ cluster that account maximally for slightly more than one spin/molecule. In addition, at temperatures below 20 K, dithionite-reduced samples exhibit broad and complex features, to high and low field of the [2Fe-2S]1+ signal, that are attributable to a spin coupled [4Fe-4S]1+ cluster. Preliminary attempts to quantify the signals indicate that the [4Fe-4S] cluster is present in an approximate 1:1 stoichiometry with the [2Fe-2S] cluster. The observed enhancement of the spin relaxation of the [2Fe-2S]1+ cluster on dithionite reduction is attributed to spin-spin interaction between the S = 1/2, reduced tetranuclear and binuclear clusters.
Dithionite↗
Electron paramagnetic resonance studies of mammalian succinate dehydrogenase. Detection of the tetranuclear cluster S2.
Electron paramagnetic resonance studies of Complex II from the mitochondrial respiratory chain and soluble preparations of succinate dehydrogenase have, for the first time, identified a signal arising from a [4Fe-4S]1+ cluster, S2, in dithionite-reduced samples. Redox titrations, monitored by electron paramagnetic resonance spectroscopy demonstrate that this signal appears at the same midpoint potential as the enhancement of the spin relaxation properties of the [2Fe-2S]1+ center, S1, in both Complex II and reconstitutively active soluble enzyme. The results complement recent magnetic circular dichroism studies of succinate dehydrogenase (Johnson, M. K., Morningstar, J. E., Bennett, D. E., Ackrell, B. A. C., and Kearney, E. B. (1985) J. Biol. Chem. 260, 7368-7378) which assigned cluster S2 as a [4Fe-4S]2+,1+ center and provide evidence for spin interaction between the paramagnetic reduced forms of centers S1 and S2.
Animals↗
Magnetic circular dichroism studies of succinate dehydrogenase. Evidence for [2Fe-2S], [3Fe-xS], and [4Fe-4S] centers in reconstitutively active enzyme.
Reconstitutively active and inactive succinate dehydrogenase have been investigated by low temperature magnetic circular dichroism (MCD) and EPR spectroscopy and room temperature CD and absorption spectroscopy. Reconstitutively active succinate dehydrogenase is found to contain three spectroscopically distinct Fe-S clusters: S1, S2, and S3. In agreement with previous studies, MCD and CD spectroscopy confirm that center S1 is a succinate-reducible [2Fe-2S]2+,1+ center. The MCD characteristics of center S2 identify it as a dithionite-reducible [4Fe-4S]2+,1+ similar to those in bacterial ferredoxins. EPR power saturation studies and the weakness of the EPR signal from reduced S2 indicate that there is a weak magnetic interaction between centers S1 and S2 in their paramagnetic, S = 1/2, reduced states. Center S3 is identified both by the form of the MCD spectrum and the characteristic magnetization behavior as a reduced [3Fe-xS] center in both succinate- and dithionite-reduced reconstitutively active succinate dehydrogenase. Arguments are presented in favor of centers S2 and S3 being separate centers rather than interconversion products of the same cluster. Reconstitutively inactive succinate dehydrogenase is found to be deficient in center S3. These results resolve many of the controversies concerning the Fe-S cluster content of succinate dehydrogenase and reconcile published EPR data with analytical and core extrusion studies. Moreover, they indicate that center S3 is a necessary requirement for reconstitutive activity and suggest that it is able to sustain ubiquinone reductase activity as a [3Fe-xS] center.
Animals↗
Interaction of the four stereoisomers of soman (pinacolyl methylphosphonofluoridate) with acetylcholinesterase and neuropathy target esterase of hen brain.
Dilute solutions in cold dry ethyl acetate of 98-100% pure specimens of each of the four stereoisomers of soman were tested against enzymes in hen brain homogenate at 37 degrees and pH 8.0. Rate constants for progressive inhibition of acetylcholinesterase were 10(7)-10(8)/mole/min for both P(-) isomers and less than 10(5) for both P(+) isomers. All isomers inhibited neuropathy target esterase non-progressively to some degree. Rate constants for progressive inhibition of neuropathy target esterase were 2.7-3.8 X 10(5)/mole/min for C(-) P(+) and 2-6 X 10(4) for the others. Forced reactivation by KF was 90% initially and aging was slow in each case. Spontaneous reactivation of inhibited neuropathy target esterase was substantial during 18 hr for both P(-) isomers but not for P(+). By comparison of rate constants for the two enzymes we predict that pure P(+) isomers may cause delayed neuropathy in hens dosed at about unprotected LD50: prophylaxis and therapy against acute cholinergic effects would have to raise LD50 1000-fold before birds could tolerate potentially neuropathic doses of P(-) isomers.
Acetylcholinesterase↗