Neuroleptic malignant syndrome in a child treated with metoclopramide for chemotherapy-related nausea.
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Child, Preschool↗
Biomedical subjects
T A Kent
Publications and source records attributed to T A Kent.
Blood platelet uptake of serotonin in episodic aggression: correlation with red blood cell proton T1 and impulsivity.
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Adolescent↗
SQUID measurement of metalloprotein magnetization. New methods applied to the nitrogenase proteins.
New techniques have been developed to exploit the sensitivity of a commercial SQUID susceptometer in the study of the magnetization of metalloproteins. Previous studies have ignored both the slow relaxation (hours) of spin I = 1/2 nuclei and residual ferromagnetic impurities in sample holders. These potential sources of noise were at or below the sensitivity of previous instruments. With these noise sources under control, one can now decrease the protein concentration by a factor of ten. In addition careful characterization of the frozen magnetization sample, including the use of a multi-instrument holder for combined study of the magnetization sample with Mössbauer spectroscopy, is required for reliable interpretation of the data in the face of paramagnetic impurities common to metalloprotein samples. Many previous magnetic studies of metalloproteins have been carried out in the Curie region. Saturation magnetization studies down to 1.8 K and up to 5 T can determine zero-field splitting parameters in addition to the spin and exchange coupling parameters measured in previous studies at lower fields and higher temperatures. Applications of these techniques to the study of the nitrogenase proteins of Azotobacter vinelandii are presented as examples.
Azotobacter↗
Amitriptyline normalizes tetrabenazine-induced changes in cerebral microcirculation.
The cerebromicrocirculation in the tetrabenzaine (TBZ) model of depression has been found to be abnormal with respect to (1) responsiveness of cerebral blood flow to increases in arterial CO2 content and (2) the effective permeability of the blood-brain barrier to water. Development of these abnormalities temporally paralleled the behavioral disturbances and catecholamine depletion induced by TBZ. These TBZ-induced changes occurred globally throughout the brain, being apparent in the forebrain, cerebellum, and medulla-pons. Pretreatment with the antidepressant amitriptyline prevented both behavioral and physiological effects of TBZ, whereas amitriptyline administered after TBZ was less effective. The results suggest that an important action of tricyclic antidepressants may be cerebromicrocirculatory effects.
Amitriptyline↗
Individual differences in state-dependent retrieval effects of alcohol intoxication.
Individual differences in susceptibility to the state-dependent retrieval (SDR) effects from alcohol in twelve subjects was tested on repeated occasions. There was a threefold variability in frequency of SDR among the subjects. A significant positive correlation between frequency of SDR and history of blackouts and heavy drinking was found.
Adult↗
Effect of hyperbaric oxygen on the blood: cerebrospinal fluid transfer of tobramycin.
The blood:cerebrospinal fluid (CSF) transfer of the aminoglycoside antibiotic, tobramycin (TOB) was assessed in rabbits. The CSF:blood ratio of TOB, 90 min after a subcutaneous injection, was approximately 1:100 as measured by an agar disc diffusion bioassay. Hyperbaric oxygen therapy at a pressure of 3 ATA caused a slight, non-significant, increase in this ratio, while high dose carbon dioxide, a treatment known to damage the blood:brain barrier, more than doubled this ratio. Thus, hyperbaric oxygen has no significant effect on CSF concentration of TOB in rabbits with intact meninges.
Animals↗
Mössbauer, EPR, and magnetization studies of the Azotobacter vinelandii Fe protein. Evidence for a [4Fe-4S]1+ cluster with spin S = 3/2.
We have studied the Fe protein (Av2) of the Azotobacter vinelandii nitrogenase system with Mössbauer and EPR spectroscopies and magnetic susceptometry. In the oxidized state the protein exhibits Mössbauer spectra typical of diamagnetic [4Fe-4S]2+ clusters. Addition of Mg.ATP or Mg.ADP causes a pronounced decline in the quadrupole splitting of the Mössbauer spectra of the oxidized protein. Our studies show that reduced Av2 in the native state is heterogeneous. Approximately half of the molecules contain a [4Fe-4S]1+ cluster with electronic spin S = 1/2 and half contain a [4Fe-4S]1+ cluster with spin S = 3/2. The former yields the characteristic g = 1.94 EPR signal whereas the latter exhibits signals around g = 5. The magnetization of reduced Av2 is dominated by the spin S = 3/2 form of its [4Fe-4S]1+ clusters. These results explain a long standing puzzle, namely why the integrated spin intensity of the g = 1.94 EPR signal is substantially less than 1 spin/4 Fe atoms. In 50% ethylene glycol, 90% of the clusters are in the spin S = 1/2 form whereas, in 0.4 M urea, 85% are in the S = 3/2 form. In 0.4 M urea, the EPR spectrum of reduced Av2 exhibits well defined resonances at g = 5.8 and 5.15, which we assign to the S = 3/2 system. The EPR and Mössbauer studies yield a zero-field splitting of 2D approximately equal to -5 cm-1 for this S = 3/2 state.
Azotobacter↗
Effect of CO2 on a brain extracellular space marker and evidence of its neuronal modulation.
Increases in inspired CO2 consistently altered the local concentration of the brain extracellular space marker alpha-naphthalene sulfonate (alpha-NS) as measured with ion-selective micropipettes in the rat thalamus. Stereotaxic injection of lidocaine in the region of the locus coeruleus attenuated this effect of CO2, and amitriptyline, a tricyclic anti-depressant and amine reuptake inhibitor, potentiated the effect. These results suggest that metabolic demand, as mimicked here by the addition of CO2, alters the fluid environment of the brain and central noradrenergic mechanisms may modulate this response.
Amitriptyline↗
Mössbauer studies of aconitase. Substrate and inhibitor binding, reaction intermediates, and hyperfine interactions of reduced 3Fe and 4Fe clusters.
Active beef heart aconitase contains a [4Fe-4S] cluster. One iron of the cluster, Fea, is labile and can be removed easily by oxidation in air to yield the [3Fe-4S]1+ cluster of inactive aconitase. We have previously shown that substrate binds to Fea. We have continued our Mössbauer studies by further investigating the active and inactive forms of the enzyme. When active aconitase, [4Fe-4S]2+, is mixed with substrate, two species (substrates or intermediates bound to Fea) labeled S1 and S2 are obtained. With the nitroanalogs of citrate and isocitrate, thought to be transition state analogs, and fluorocitrate, species S2, but not S1, is observed, suggesting that S2 represents a carbanion transition state complex. We have prepared Mössbauer samples by rapid mix/rapid freeze techniques. Using either citrate, isocitrate or cis-aconitate, the natural substrates, we have been able to detect at 0 degree C reaction intermediates in the 5-35 ms time range and, studying enzyme substrate interactions at subzero temperatures in a water/methanol/ethylene glycol solvent, we have observed new species when substrates were added at -60 degrees C. Details of these experiments are given, although in neither case can unique interpretations be offered at this time. We have also investigated reduced active aconitase ([4Fe-4S]1+; EPR at g = 1.94) in the presence of substrate with material selectively enriched with 57Fe in either Fea or the other three cluster sites. The spectra were analyzed with a spin Hamiltonian, and the results are discussed and interpreted in terms of three inequivalent Fe sites in the cluster. Finally, we have studied enzyme containing the reduced [3Fe-4S]0 cluster. There is no indication that citrate binds to the 3Fe cluster, and since no significant activity was observed, we conclude that aconitase containing a 3Fe cluster is not active in either oxidation state.
Aconitate Hydratase↗
A modified method for the simultaneous determination of regional single-transit brain extraction of diffusion-limited compounds and cerebral blood flow: utilization of non-invasive measurement of transit time.
A dual-label radioisotope method to measure regional cerebral blood flow (CBF) with [14C]butanol and the single-transit brain extraction of [3H]water (Ew) was modified to permit concomittant measurement of the exit time through the cerebrovasculature of a bolus (BET) of 51chromium (51Cr)-labeled ethylenediaminetetraacetic acid (EDTA) in rodents. First, [51Cr]EDTA was injected intravenously via the femoral vein and its transit through cerebrovasculature determined by external gamma counting. The BET measurement was then used to determine the optimum time for animal sacrifice for subsequent measurement of CBF and Ew to minimize both intravascular contamination and washout of butanol and water. This procedure resulted in higher CBF at moderate hypercapnia and slightly lower Ew as a function of arterial CO2 content than previously found using a fixed interval for sacrifice.
1-Butanol↗
Blood platelet uptake of serotonin in men alcoholics.
Blood platelet uptake of 3H-serotonin (5HT uptake) was determined in a group of inpatient alcoholics who had just undergone detoxication (N = 19) and an age-matched group of nonalcoholic controls (N = 19). Mean 5HT uptake was 18% lower in the alcoholics (p less than .01). A 3-min 5HT uptake of less than 6 pmoles/10(7) platelets identified 58% of the alcoholics, but only 15% of the controls had a 5HT uptake lower than this value. These results support the hypothesis that serotonergic dysfunction may exist in alcoholics.
Adult↗
Evidence for the formation of a linear [3Fe-4S] cluster in partially unfolded aconitase.
Beef heart aconitase, as isolated under aerobic conditions, is inactive and contains a [3Fe-4S]1+ cluster. On incubation at pH greater than 9.5 (or treatment with 4-8 M urea) the color of the protein changes from brown to purple. This purple form is stable and can be converted back in good yield to the active [4Fe-4S]2+ form by reduction in the presence of iron. Active aconitase is converted to the purple form at alkaline pH only after oxidative inactivation. The Fe/S2- ratio of purple aconitase is 0.8, indicating the presence of [3Fe-4S] clusters. The number of SH groups readily reacting with 5,5'-dithiobis(2-nitrobenzoic acid) is increased from approximately 1 in the enzyme as isolated to 7-8 in the purple form, indicating a partial unfolding of the protein. On conversion of inactive aconitase to the purple form, the EPR signal at g = 2.01 (S = 1/2) is replaced by signals at g = 4.3 and 9.6 (S = 5/2). Mössbauer spectroscopy shows that purple aconitase has high-spin ferric ions, each residing in a tetrahedral environment of sulfur atoms. The three iron sites are exchange-coupled to yield a ground state with S = 5/2. Analysis of the data within a spin coupling model shows that J13 congruent to J23 and 2 J12 less than J13, where the Jik describe the antiferromagnetic (J greater than 0) exchange interactions among the three iron pairs. Comparison of our data with those reported for synthetic Fe-S clusters (Hagen, K. S., Watson, A. D., and Holm, R. H., (1983) J. Am. Chem. Soc. 105, 3905-3913) shows that purple aconitase contains a linear [3Fe-4S]1+ cluster, a structural isomer of the S = 1/2 cluster of inactive aconitase. Our studies also show that protein-bound [2Fe-2S] clusters can be generated under conditions where partial unfolding of the protein occurs.
Aconitate Hydratase↗
Mössbauer, EPR, and optical studies of the P-460 center of hydroxylamine oxidoreductase from Nitrosomonas. A ferrous heme with an unusually large quadrupole splitting.
Hydroxylamine oxidoreductase from Nitrosomonas europeae catalyzes the oxidative conversion of NH2OH to NO-2. The enzyme, Mr = 220,000, has an (alpha beta)3 subunit structure with each alpha beta subunit containing 7-8 c-type hemes and one unusual prosthetic group, termed P-460. The P-460 is also found in a Mr approximately equal to 17,000 protein (P-460 fragment). Mössbauer spectra of the reduced P-460 groups, in hydroxylamine oxidoreductase and the fragment, exhibit nearly identical quadrupole doublets with an unusually large splitting, delta EQ = 4.21 mm/s (no ferrous heme protein is known with delta EQ greater than 2.75 mm/s). The observed isomer shift, delta = 0.96 mm/s at 4.2 K, shows that the P-460 iron is high spin ferrous. Treatment of oxidized hydroxylamine oxidoreductase with H2O2 followed by reduction or exposure of the native sample to CO led to the disappearance of both the characteristic 460 nm absorption band (epsilon = 89 mM-1 cm-1) and the delta EQ = 4.21 mm/s doublet. The iron of the oxidized P-460 fragment is high spin ferric, with Mössbauer and EPR parameters very similar to those of metmyoglobin. Optical spectra of the reduced P-460 fragment show long wavelength bands at 650 and 688 nm which are sensitive to treatment of the fragment with reagents which react with P-460. These bands were, however, not detected in hydroxylamine oxidoreductase. The spectroscopic and chemical evidence obtained to date suggests strongly that the P-460 iron resides in a heme-like macrocycle although the presumed porphyrin must have some unusual features.
Electron Spin Resonance Spectroscopy↗
Brevibacterium fuscum protocatechuate 3,4-dioxygenase. Purification, crystallization, and characterization.
A protocatechuate 3,4-dioxygenase with exceptionally sharp spectral features and a new subunit composition has been purified and crystallized from the Gram-positive organism Brevibacterium fuscum. EPR spectra show that the catalytically essential Fe3+ resides in a site of almost the maximal rhombicity (E/D = 0.333 +/- 0.003). The spectral line widths (1.4 milliTesla at g = 9.67) are the smallest reported for any biological high spin Fe3+ complex and suggest that the enzyme is quite homogeneous in the vicinity of the Fe site. The same conclusion is drawn from Mössbauer spectra measured with enzyme prepared from cells cultured in 57Fe-enriched media as well as from resonance Raman and optical spectra. In contrast, EPR and Mössbauer spectra of the anaerobic complex with protocatechuate (PCA) are complex and demonstrate that multiple species with markedly different electronic symmetries and both positive and negative zero field splittings are present. The Mr = 315,000 enzyme has a composition of (alpha beta Fe)5 (Mr(alpha) = 22,500; Mr (beta) = 40,000). Amino acid analysis shows that neither subunit contains cysteine, thus eliminating this amino acid as a possible Fe ligand. The general features of the structure, spectra, and catalyzed reaction of this enzyme appear to be very similar to those of protocatechuate 3,4-dioxygenase isolated from Gram-negative organisms. However, the kinetic parameters (Km(PCA) = 125 microM, Km(O2) = 800 microM, turnover number = 25,000 min-1 at infinite PCA and O2 concentrations) are 5- to 50-fold higher. The sharp spectra and the kinetic properties facilitate mechanistic studies described in this and the following reports.
Amino Acids↗
Purification and characterization of the Rieske iron-sulfur protein from Thermus thermophilus. Evidence for a [2Fe-2S] cluster having non-cysteine ligands.
We have purified the Rieske iron-sulfur protein from Thermus thermophilus. Chemical analyses show that the protein contains iron, labile sulfide, and cysteine in equimolar concentrations, four of each for Mr approximately 20,000. The oxidized and reduced form of the protein have been characterized by optical, EPR, CD, magnetic CD and Mössbauer spectroscopies. Our data suggest the presence of a unique iron-sulfur center. Mössbauer studies of the oxidized and reduced protein demonstrate unambiguously that the protein contains clusters with [2Fe-2S] cores. The iron analyses and the Mössbauer data, taken together, suggest that the protein has two [2Fe-2S] clusters. This is supported by the observation that two electrons are required for complete reduction of the protein and that the g = 1.94-type signal of the reduced protein has a spin concentration of one spin (S = 1/2) per 2Fe. Within the excellent resolution of the Mössbauer and EPR data, the two clusters are identical. Our results thus suggest that each [2Fe-2S] cluster is coordinated by at most two cysteine residues. The Mössbauer spectra of the reduced protein were analyzed with an S = 1/2 spin Hamiltonian. The hyperfine parameters obtained are very similar to those reported for putidaredoxin. The main difference is that the Rieske protein exhibits an increased isomer shift at the Fe2+ site, suggesting that non-cysteine ligands are coordinated to the site that becomes reduced to Fe2+ upon reduction. A comparison of our data with those reported for various NADH-dependent dioxygenases suggest that these enzymes contain a Rieske-type [2Fe-2S] center.
Amino Acids↗
Cerebromicrocirculatory defects in animal model of depression.
In the tetrabenazine (TBZ) model of depression, the cerebromicrocirculation was discovered to respond abnormally to metabolic demand as mimicked by the administration of CO2. Altered responsivity of cerebral blood flow and effective permeability of the blood--brain barrier to changes in PaCO2 were found. These physiologic defects coincided temporally with TBZ-induced depletion of central norepinephrine and dopamine and with the development of the behavioral effects of TBZ (the end points used to test the antidepressant potential of experimental drugs). Pretreatment with amitriptyline (a standard antidepressant and amine reuptake inhibitor) prevented the development of these TBZ-induced abnormalities in the cerebromicrocirculation, just as it prevented the behavioral effects.
Amitriptyline↗
Cerebral blood flow and cerebrovascular permeability in an inescapable shock (learned helplessness) animal model of depression.
The effects of a purported animal model of depression (inescapable shock, IS) was tested on: (a) escape behavior, (b) regional brain levels of norepinephrine (NE), serotonin (5-HT), and dopamine, and (c), the response of the cerebromicrovasculature to metabolic demand as mimicked by manipulation of arterial CO2 content (PaCO2). Multidisciplinary research has implicated central biogenic amines in the regulation of cerebromicrocirculation. IS treatment resulted in increased escape latency and lowered levels of NE and 5-HT in the locus coeruleus but not in terminal fields in distant regions. This treatment also did not alter cerebral blood flow or capillary permeability in distant regions when compared with control rats. Thus, the discrete changes in NE and 5-HT in locus coeruleus induced by IS treatment is not reflected in changes in cerebral blood flow and the effective permeability of the blood-brain barrier.
Animals↗
Mössbauer studies of iron proteins.
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Animals↗