Substitution of tyrosine for the proximal histidine ligand to the heme of prostaglandin endoperoxide synthase 2: implications for the mechanism of cyclooxygenase activation and catalysis.

Prostaglandin H(2) synthesis by prostaglandin endoperoxide synthase (PGHS) requires the heme-dependent activation of the protein's cyclooxygenase activity. The PGHS heme participates in cyclooxygenase activation by accepting an electron from Tyr385 located in the cyclooxygenase active site. Two mechanisms have been proposed for the oxidation of Tyr385 by the heme iron: (1) ferric enzyme oxidizes a hydroperoxide activator and the incipient peroxyl radical oxidizes Tyr385, or (2) ferric enzyme reduces a hydroperoxide activator and the incipient ferryl-oxo heme oxidizes Tyr385. The participation of ferrous PGHS in cyclooxygenase activation was evaluated by determining the reduction potential of PGHS-2. Under all conditions tested, this potential (<-135 mV) was well below that required for reactions leading to cyclooxygenase activation. Substitution of the proximal heme ligand, His388, with tyrosine was used as a mechanistic probe of cyclooxygenase activation. His388Tyr PGHS-2, expressed in insect cells and purified to homogeneity, retained cyclooxygenase activity but its peroxidase activity was diminished more than 300-fold. Concordant with this poor peroxidase activity, an extensive lag in His388Tyr cyclooxygenase activity was observed. Addition of hydroperoxides resulted in a concentration-dependent decrease in lag time consistent with each peroxide's ability to act as a His388Tyr peroxidase substrate. However, hydroperoxide treatment had no effect on the maximal rate of arachidonate oxygenation. These data imply that the ferryl-oxo intermediates of peroxidase catalysis, but not the Fe(III)/Fe(II) couple of PGHS, are essential for cyclooxygenase activation. In addition, our findings are strongly supportive of a branched-chain mechanism of cyclooxygenase catalysis in which one activation event leads to many cyclooxygenase turnovers.

Structural insights into the stereochemistry of the cyclooxygenase reaction.

Cyclooxygenases are bifunctional enzymes that catalyse the first committed step in the synthesis of prostaglandins, thromboxanes and other eicosanoids. The two known cyclooxygenases isoforms share a high degree of amino-acid sequence similarity, structural topology and an identical catalytic mechanism. Cyclooxygenase enzymes catalyse two sequential reactions in spatially distinct, but mechanistically coupled active sites. The initial cyclooxygenase reaction converts arachidonic acid (which is achiral) to prostaglandin G2 (which has five chiral centres). The subsequent peroxidase reaction reduces prostaglandin G2 to prostaglandin H2. Here we report the co-crystal structures of murine apo-cyclooxygenase-2 in complex with arachidonic acid and prostaglandin. These structures suggest the molecular basis for the stereospecificity of prostaglandin G2 synthesis.

Spatial requirements for 15-(R)-hydroxy-5Z,8Z,11Z, 13E-eicosatetraenoic acid synthesis within the cyclooxygenase active site of murine COX-2. Why acetylated COX-1 does not synthesize 15-(R)-hete.

The two isoforms of cyclooxygenase, COX-1 and COX-2, are acetylated by aspirin at Ser-530 and Ser-516, respectively, in the cyclooxygenase active site. Acetylated COX-2 is essentially a lipoxygenase, making 15-(R)-hydroxyeicosatetraenoic acid (15-HETE) and 11-(R)-hydroxyeicosatetraenoic acid (11-HETE), whereas acetylated COX-1 is unable to oxidize arachidonic acid to any products. Because the COX isoforms are structurally similar and share approximately 60% amino acid identity, we postulated that differences within the cyclooxygenase active sites must account for the inability of acetylated COX-1 to make 11- and 15-HETE. Residues Val-434, Arg-513, and Val-523 were predicted by comparison of the COX-1 and -2 crystal structures to account for spatial and flexibility differences observed between the COX isoforms. Site-directed mutagenesis of Val-434, Arg-513, and Val-523 in mouse COX-2 to their COX-1 equivalents resulted in abrogation of 11- and 15-HETE production after aspirin treatment, confirming the hypothesis that these residues are the major isoform selectivity determinants regulating HETE production. The ability of aspirin-treated R513H mCOX-2 to make 15-HETE, although in reduced amounts, indicates that this residue is not an alternate binding site for the carboxylate of arachidonate and that it is not the only specificity determinant regulating HETE production. Further experiments were undertaken to ascertain whether the steric bulk imparted by the acetyl moiety on Ser-530 prevented the omega-end of arachidonic acid from binding within the top channel cavity in mCOX-2. Site-directed mutagenesis was performed to change Val-228, which resides at the junction of the main cyclooxygenase channel and the top channel, and Gly-533, which is in the top channel. Both V228F and G533A produced wild type-like product profiles, but, upon acetylation, neither was able to make HETE products. This suggests that arachidonic acid orientates in a L-shaped binding configuration in the production of both prostaglandin and HETE products.

Effects of nitric oxide and nitric oxide-derived species on prostaglandin endoperoxide synthase and prostaglandin biosynthesis.

Prostaglandins and NO. are important mediators of inflammation and other physiological and pathophysiological processes. Continuous production of these molecules in chronic inflammatory conditions has been linked to development of autoimmune disorders, coronary artery disease, and cancer. There is mounting evidence for a biological relationship between prostanoid biosynthesis and NO. biosynthesis. Upon stimulation, many cells express high levels of nitric oxide synthase (NOS) and prostaglandin endoperoxide synthase (PGHS). There are reports of stimulation of prostaglandin biosynthesis in these cells by direct interaction between NO. and PGHS, but this is not universally observed. Clarification of the role of NO. in PGHS catalysis has been attempted by examining NO. interactions with purified PGHS, including binding to its heme prosthetic group, cysteines, and tyrosyl radicals. However, a clear picture of the mechanism of PGHS stimulation by NO. has not yet emerged. Available studies suggest that NO. may only be a precursor to the molecule that interacts with PGHS. Peroxynitrite (from O2.-+NO.) reacts directly with PGHS to activate prostaglandin synthesis. Furthermore, removal of O2.- from RAW 267.4 cells that produce NO. and PGHS inhibits prostaglandin biosynthesis to the same extent as NOS inhibitors. This interaction between reactive nitrogen species and PGHS may provide new approaches to the control of inflammation in acute and chronic settings.

Nitric oxide trapping of tyrosyl radicals generated during prostaglandin endoperoxide synthase turnover. Detection of the radical derivative of tyrosine 385.

Tyrosyl radicals have been detected during turnover of prostaglandin endoperoxide H synthase (PGHS), and they are speculated to participate in cyclooxygenase catalysis. Spectroscopic approaches to elucidate the identity of the radicals have not been definitive, so we have attempted to trap the radical(s) with nitric oxide (NO). NO quenched the EPR signal generated by reaction of purified ram seminal vesicle PGHS with arachidonic acid, suggesting that NO coupled with a tyrosyl radical to form inter alia nitrosocyclohexadienone. Subsequent formation of nitrotyrosine was detected by Western blotting of PGHS incubated with NO and arachidonic acid or organic hydroperoxides using an antibody against nitrotyrosine. Both arachidonic acid and NO were required to form nitrotyrosine, and tyrosine nitration was blocked by the PGHS inhibitor indomethacin. The presence of superoxide dismutase had no effect on nitration, indicating that peroxynitrite was not the nitrating agent. To identify which tyrosines were nitrated, PGHS was digested with trypsin, and the resulting peptides were separated by high pressure liquid chromatography and monitored with a diode array detector. A single peptide was detected that exhibited a spectrum consistent with the presence of nitrotyrosine. Consistent with Western blotting results, both NO and arachidonic acid were required to observe nitration of this peptide, and its formation was blocked by the PGHS inhibitor indomethacin. Peptide sequencing indicated that the modified residue was tyrosine 385, the source of the putative catalytically active tyrosyl radical.

Roles of efficient substrates in enhancement of peroxidase-catalyzed oxidations.

Efficient peroxidase substrates may have a critical role in the oxidation of secondary compounds by peroxidases. Hydrazines are often oxidized slowly by peroxidases due, in part, to hydrazine-dependent inactivation of these enzymes. Peroxidase-catalyzed oxidation of hydrazines may be dramatically affected by an efficient peroxidase substrate. We investigated this hypothesis in a model system using the well-known peroxidase substrate chlorpromazine (CPZ) and the hydrazine derivative isoniazid. CPZ stimulated isoniazid oxidation as measured by nitroblue tetrazolium (NBT) reduction and O2 consumption. The kinetics of isoniazid and CPZ oxidation by horseradish peroxidase (HRP) in the presence of both compounds suggested CPZ was acting as an electron transfer mediator between HRP and isoniazid. Indeed, CPZ.+, the product of CPZ oxidation by HRP, was able to oxidize isoniazid. The rate constant for this pH-dependent reaction was (2.6 +/- 0.1) x 10(4) M-1 s-1 at pH 4.5. In the absence of CPZ, isoniazid-dependent irreversible inactivation of HRP was observed. The inactivation process involved the formation of compound III followed by accumulation of irreversibly inactivated HRP. CPZ completely inhibited inactivation. Thus, by acting as a redox mediator and preventing HRP inactivation, CPZ stimulated isoniazid oxidation by several orders of magnitude. Similarly, other efficient peroxidase substrates, such as phenol and tyrosine, were also able to dramatically stimulate isoniazid oxidation by HRP. We suggest that the presence of efficient peroxidase substrates may potentiate the activation of isoniazid and other hydrazines. As such, these substrates may have a vital role in the pharmacological and toxicological properties of hydrazines and other compounds.

Free radicals produced during the oxidation of hydrazines by hypochlorous acid.

Hypochlorous acid (HOCl) derived from activated neutrophils and monocytes has been implicated in the activation of hydrazine-containing drugs to toxic intermediates. However, reactive intermediates formed during the reaction between HOCl and these drugs have not been identified. We investigated the oxidation of the hydrazine derivatives isoniazid, iproniazid, and hydralazine by HOCl. The reaction between HOCl and all three hydrazines resulted in O2 consumption, indicating that free radicals were produced, but the rate and extent of O2 consumption were different for each hydrazine. Moreover, reduction of nitroblue tetrazolium (NBT) was observed only during the reaction between HOCl and isoniazid, suggesting that different radical species may be produced from HOCl reaction with each hydrazine. The oxidation of iproniazid by HOCl in the presence of the radical trap 5,5-dimethyl-1-pyrroline N-oxide (DMPO) resulted in the formation of a carbon-centered radical adduct. In contrast, the reaction between HOCl and hydralazine resulted in the formation of a nitrogen-centered DMPO radical adduct. The oxidation of isoniazid by HOCl resulted in the formation of two oxygen-centered radical adducts, DMPO-OOH and DMPO-OH. Myeloperoxidase-catalyzed oxidation of these hydrazines in the presence of Cl- and H2O2 produced radical species that were identical to those observed with HOCl. Thus, some of the toxic side effects of these drugs may be the result of the production of free-radical intermediates from reaction with neutrophil-derived oxidants, such as HOCl. The types of radicals produced and the consequences of generating these reactive species are discussed.

Redox mediation in the peroxidase-catalyzed oxidation of aminopyrine: possible implications for drug-drug interactions.

Many drugs, industrial pollutants, and other xenobiotics are known to be oxidized by peroxidases to potentially harmful free-radical intermediates. We have examined the possibility that certain compounds, acting as efficient peroxidase substrates, may stimulate the formation of reactive free radicals by acting as mediators of electron transfer reactions (redox mediators). To explore this hypothesis, we have investigated the interaction of two well-known peroxidase substrates, chlorpromazine and aminopyrine. As shown by ESR and UV-visible spectroscopy, chlorpromazine radical was able to oxidize aminopyrine to aminopyrine cation radical. The rate constant for this rapid, pH-dependent, reaction was estimated to be 1 x 10(7) M-1 s-1 at pH 4.5. Transient-state and steady-state kinetic studies both showed that rate constants for chlorpromazine oxidation to its cation radical by horseradish peroxidase (HRP) were about 100-fold greater than for the corresponding HRP-catalyzed oxidation of aminopyrine to its cation radical. When both aminopyrine and chlorpromazine were present with HRP and H2O2, aminopyrine cation radical formation was stimulated 100-fold. Concomitantly, the accumulation of chlorpromazine cation radical was completely inhibited in the presence of aminopyrine. Similar results were obtained when lactoperoxidase, myeloperoxidase, or the myeloperoxidase mimic HOCl were substituted for HRP. These data suggest that chlorpromazine can act as a redox mediator for peroxidase-catalyzed oxidation of aminopyrine and other chemicals. We suggest that some peroxidase substrates, acting as redox mediators, may stimulate the production of toxic free-radical intermediates from various drugs and other xenobiotics. As such, this may have implications for a number of adverse effects caused by these xenobiotic chemicals.

Determination of rate constants for rapid peroxidase reactions.

We describe an improved enzyme-monitored stopped-flow method for determining rate constants for peroxidase reactions that are too rapid to measure by conventional pseudo-first-order methods. Ascorbate will reduce many substrate radicals as rapidly as they are generated by a peroxidase. This ensures a constant substrate concentration during the peroxidase reaction. We investigated the reactions of horseradish peroxidase compound I (HRP-I) with three standard substrates (chlorpromazine (CPZ), 2,2'-azino-bis[3-ethylbenzthiazoline-6-sulfonic acid] (ABTS), and p-methoxyphenol) in the presence and absence of ascorbate. The rate of reaction of CPZ with HRP-I is slow enough that it can be measured using pseudo-first-order conditions maintained by a minimum 10-fold excess of CPZ, or by the addition of ascorbate at very low CPZ concentrations. The same rate constant was obtained with either method. The rate of reaction of ABTS with HRP-I at lower pH (5.0-3.3) is rapid; consequently, we were unable to obtain rate constants using concentrations of ABTS which constitute pseudo-first-order conditions. However, using much lower ABTS concentrations with the addition of ascorbate, we obtained rate constants that ranged from 5 x 10(7) to 8 x 10(8) M-1 s-1. Hence, ascorbate provides a simplified way to maintain pseudo-first-order conditions for fast peroxidase reactions even at low substrate concentrations.

Evidence for veratryl alcohol as a redox mediator in lignin peroxidase-catalyzed oxidation.

We have examined the hypothesis that veratryl alcohol (VA) may act as a redox mediator in lignin peroxidase (LiP)-catalyzed oxidations. The oxidation of chlorpromazine (CPZ) by this system was used to evaluate this hypothesis. Chlorpromazine can be oxidized by one electron to form a stable cation radical (CPZ.+). This cation radical can be oxidized by another electron to the sulfoxide (CPZSO). These oxidation steps are easily monitored, making CPZ a useful chemical to investigate redox mediation by VA. Lignin peroxidase oxidized CPZ to CPZ.+ whether or not VA was present. The inclusion of VA, however, stimulated CPZ oxidation to CPZ.+ and subsequent oxidation of CPZ.+ to CPZSO. In the absence of VA, the initial rates of CPZ oxidation by LiP were CPZ concentration-dependent. However, when saturating concentrations of VA were added, the oxidation of CPZ and CPZ.+ became independent of CPZ concentration. When the oxidation of VA to veratryl aldehyde was examined, increasing concentrations of CPZ produced a lag in veratryl aldehyde appearance proportional to the concentration of CPZ. Conversely, increasing concentrations of VA never inhibited CPZ oxidation. Transient-state kinetic studies indicated that both VA and CPZ reduced the compound I and compound II forms of LiP. However, when saturating concentrations of VA were utilized, LiP turnover was independent of CPZ concentration. We suggest these data demonstrate that VA may act as a redox mediator for the indirect oxidation of compounds by LiP.

The role of oxalate in lignin peroxidase-catalyzed reduction: protection from compound III accumulation.

Reduction may be an important step in the degradation of some highly oxidized environmental pollutants by Phanerochaete chrysosporium. Lignin peroxidases (LiP) from P. chrysosporium are able to catalyze reductive reactions using veratryl alcohol (VA) as a mediator and either oxalate or EDTA as electron donors. Reduction of oxygen to superoxide, monitored by oxygen consumption, was used as a measure of the reductive activity of LiP. In the presence of EDTA, the rate of O2 reduction catalyzed by LiP decreased with time and increasing concentrations of H2O2. When oxalate replaced EDTA, LiP-catalyzed O2 reduction did not decrease with time, and increasing concentrations of H2O2 increased the duration and extent of O2 reduction. LiP was converted to the compound III state in the presence of EDTA, H2O2, and veratryl alcohol. When oxalate replaced EDTA, compound II was observed. The importance of the veratryl alcohol cation radical (VA.+) in the conversion of LiP compound III to active enzyme has been previously examined (D.P. Barr and S.D. Aust, 1994, Arch. Biochem. Biophys. 311, 378-382). We propose that rapid reduction of VA.+ by EDTA results in accumulation of LiP compound III and the loss of activity resulting in a decrease in LiP-catalyzed reduction reactions. Oxalate is less effective in reducing the VA.+, therefore, some VA.+ remains to convert compound III to active enzyme and maintain LiP-catalyzed reduction reactions. Thus oxalate, a normal secondary metabolite of P. chrysosporium, is a suitable candidate for mediating reduction reactions by LiP in vivo.

Shifts in diagnostic frequencies of schizophrenia and major affective disorders at six North American psychiatric hospitals, 1972-1988.

OBJECTIVE: This study tested the impression that there have been significant shifts in the relative diagnostic frequencies of schizophrenia and major affective disorders. METHOD: Data on discharge diagnoses from 1972 to 1988 were gathered from six North American psychiatric teaching hospitals (data from one extended through 1991), and rates for schizophrenia and major mood disorders were evaluated. RESULTS: Total annual discharges increased by 6.6% during the study period. Large reciprocal shifts in the frequencies of diagnoses of schizophrenia and major affective disorders were found; schizoaffective disorder was a minor diagnosis. Beginning in the early 1970s, a gradual increase in the frequency of diagnoses of major affective disorders at all sites was accompanied by a corresponding decrease in diagnoses of schizophrenia at five of the six centers. Schizophrenia diagnoses decreased from a peak of 27% in 1976 to 9% in 1989 (a threefold decrease), and diagnoses of major affective disorders rose from a low of 10% in 1972 to 44% in 1990 (a fourfold increase). CONCLUSIONS: Several forces may have influenced these changes. 1) DSM-III narrowed the definition of schizophrenia and broadened the category of major affective disorders. 2) Treatment-oriented diagnostic bias associated with the availability of lithium and other mood-altering agents may have encouraged consideration of affective disorders. 3) Economic and social forces, including better third-party reimbursement rates, may have favored affective diagnoses. 4) True increases in the incidence of affective disorders may have occurred. 5) Although a real decrease in new cases of schizophrenia may have occurred, this effect was probably minor and dominated by a larger shift of such diagnoses to affective categories.

Microwave miniprep of total genomic DNA from fungi, plants, protists and animals for PCR.

A rapid procedure for the isolation of total DNA from various eukaryotic organisms is described. This method uses a common microwave oven for the alteration of cellular walls and membranes. The procedure is rapid and can be completed in as little as 15 minutes. The method has been used to isolate DNA from reptiles, plants, slime molds and other protists, and this DNA has been used for successful amplification of ribosomal genes from these organisms. The procedure is a time-saving and cost-effective process that helps to eliminate several sources of contamination that can be found in other DNA isolation procedures.

The Tridimensional Personality Questionnaire as a predictor of six-month outcome in first episode mania.

The authors administered the Tridimensional Personality Questionnaire (TPQ) near hospital discharge to 27 patients with first episode mania. All patients met DSM-III-R criteria for bipolar disorder, manic type, as assessed by the Structured Clinical Interview for DSM-III-R. Associations of TPQ scores with operationalized outcome variables were analyzed. Outcome variables included syndromic recovery at discharge and at 6 months, syndromic recurrence, and functional recovery. Patients who failed to achieve functional recovery by 6 months had significantly higher Novelty-Seeking dimensional scores at the time of hospital discharge. This association between TPQ scores and short-term outcome suggests that elevated Novelty-Seeking scores may reflect either personality characteristics that impair functional recovery or subclinical manic symptomatology that is not reflected in other symptom measures. The TPQ may provide useful prognostic measures in patients with new onset mania.

Comorbidity in psychosis at first hospitalization.

OBJECTIVE: The authors sought to determine the prevalence and effects of medical and psychiatric comorbidity on initial outcome in a group of patients experiencing a first episode of psychosis. METHOD: Patients with a first episode of psychosis who were consecutively admitted to a hospital (N = 102) were examined for the presence of psychiatric and medical disorders. Patients were given psychiatric diagnoses with the use of the Structured Clinical Interview for DSM-III-R and were rated weekly on symptom rating scales. Outcome variables at discharge were final symptom rating scale scores, length of hospitalization, and recovery on the basis of operationalized criteria. RESULTS: Comorbid diagnoses were present in 52.0% (N = 53) of the patients, and 37.7% (N = 20) had multiple comorbid diagnoses. The most common comorbid diagnosis was substance abuse. Patients with affected psychoses were significantly more likely than those with nonaffective psychoses to have a comorbid substance abuse diagnosis. Patients with psychiatric comorbidity had poorer initial outcomes, while those with medical comorbidity had fewer symptoms at discharge. CONCLUSIONS: Comorbidity is common and may be a useful predictor of the outcome of a first episode of psychosis.

Prediction of outcome in mania by mood-congruent or mood-incongruent psychotic features.

OBJECTIVE: The aim of this study was to determine the significance of mood congruence of psychotic features in mania as a predictor of outcome. METHOD: Fifty-four patients with bipolar disorder were followed prospectively for 4 years after recovery from an episode of mania with psychotic features. Assessments of residential and occupational status, interepisode symptoms, and episode recurrences were made at 6 and 48 months after recovery. Categorical outcomes were evaluated by logistic regression and recurrence risk with survival analysis. RESULTS: Mood-incongruent psychotic features during the index manic episode predicted a shorter time in remission at 4 years (hazard ratio = 2.6), and Schneiderian first-rank symptoms predicted poor residential status at 4 years (odds ratio = 20.1). CONCLUSIONS: Differentiation of mood congruence of psychotic features in mania evidently has prognostic validity and, therefore, has utility as a nosological characteristic.

Comorbidity in mania at first hospitalization.

Comorbidity was studied in 41 manic and mixed-state bipolar patients at first hospitalization. The lifetime prevalence of comorbidity was high; 21 subjects (51.2%) had at least one other psychiatric diagnosis (N = 16, 39.0%) or medical disorder (N = 9, 22.0%). Nine subjects had multiple comorbid diagnoses. Women were 2.7 times more likely to have a comorbid diagnosis. The presence of comorbidity was not associated with differences in outcome measures.

Possible affective-state dependence of the Tridimensional Personality Questionnaire in first-episode psychosis.

The authors administered the Tridimensional Personality Questionnaire (TPQ) to 61 patients with first-episode psychosis. Subjects were classified into affective states according to DSM-III-R diagnoses. TPQ scores were compared among these states and correlated with two affective symptom subscales: "mania" and "depression." Manic subjects demonstrated little variation from normative TPQ scores. Compared with findings in manic subjects, the dimensional score for Harm Avoidance was elevated in all affective groups, "worry and pessimism" was elevated in mixed-state subjects, "shyness with strangers" was elevated in depressed and nonaffective subjects, and "attachment" was lower in depressed and nonaffective subjects. The Harm Avoidance dimensional score and two subdimensional scores were positively correlated with the "depression" subscale. The Harm Avoidance dimensional and subdimensional scores showed possible affective-state dependence that may limit the utility of this instrument as a personality measure in first-episode psychosis.