Detecting changes in distortion product otoacoustic emission maps using statistical parametric mapping and random field theory.

Distortion product otoacoustic emission (DPOAE) maps collect DPOAE emissions over a broad range of frequencies and ratios. One application of DPOAE mapping could be monitoring changes in intracranial pressure (ICP) in space, where non-invasive measures of ICP are an area of interest. Data were collected in two experiments to statistically assess changes in DPOAE maps. A repeatability study where four maps per subject were collected across four weeks to establish "normal" variability in DPOAE data, and a posture study where subjects were measured supine and prone with lower body negative pressure, lower body positive pressure (LBPP), and at atmospheric pressure. DPOAE amplitude maps were analyzed using statistical parametric mapping and random field theory. Postural changes produced regional changes in the maps, specifically in the range of 5-7.5 kHz and between primary tone ratios of 1.13-1.24. These regional changes were most pronounced in the prone LBPP condition, where amplitudes were lower from baseline for the Postural Cohort than the Repeatability Cohort. Statistical parametric mapping provided a sensitive measure of regional DPOAE map changes, which may be useful clinically to monitor ICP noninvasively in individuals or for research to identify differences within in cohorts of people.

Indications of nonlinear deterministic and finite-dimensional structures in time series of brain electrical activity: dependence on recording region and brain state.

We compare dynamical properties of brain electrical activity from different recording regions and from different physiological and pathological brain states. Using the nonlinear prediction error and an estimate of an effective correlation dimension in combination with the method of iterative amplitude adjusted surrogate data, we analyze sets of electroencephalographic (EEG) time series: surface EEG recordings from healthy volunteers with eyes closed and eyes open, and intracranial EEG recordings from epilepsy patients during the seizure free interval from within and from outside the seizure generating area as well as intracranial EEG recordings of epileptic seizures. As a preanalysis step an inclusion criterion of weak stationarity was applied. Surface EEG recordings with eyes open were compatible with the surrogates' null hypothesis of a Gaussian linear stochastic process. Strongest indications of nonlinear deterministic dynamics were found for seizure activity. Results of the other sets were found to be inbetween these two extremes.

Nonlinear EEG analysis in epilepsy: its possible use for interictal focus localization, seizure anticipation, and prevention.

Several recent studies emphasize the high value of nonlinear EEG analysis particularly for improved characterization of epileptic brain states. In this review the authors report their work to increase insight into the spatial and temporal dynamics of the epileptogenic process. Specifically, they discuss possibilities for seizure anticipation, which is one of the most challenging aspects of epileptology. Although there are numerous studies exploring basic neuronal mechanisms that are likely to be associated with seizures, to date no definite information is available regarding how, when, or why a seizure occurs. Nonlinear EEG analysis now provides strong evidence that the interictal-ictal state transition is not an abrupt phenomenon. Rather, findings indicate that it is indeed possible to detect a preseizure phase. The unequivocal definition of such a state with a sufficient length would enable investigations of basic mechanisms leading to seizure initiation in humans, and development of adequate seizure prevention strategies.

Structure of eicosapentaenoic and linoleic acids in the cyclooxygenase site of prostaglandin endoperoxide H synthase-1.

Prostaglandin endoperoxide H synthases-1 and -2 (PGHSs) can oxygenate 18-22 carbon polyunsaturated fatty acids, albeit with varying efficiencies. Here we report the crystal structures of eicosapentaenoic acid (EPA, 20:5 n-3) and linoleic acid (LA, 18:2 n-6) bound in the cyclooxygenase active site of Co(3+) protoporphyrin IX-reconstituted ovine PGHS-1 (Co(3+)-oPGHS-1) and compare the effects of active site substitutions on the rates of oxygenation of EPA, LA, and arachidonic acid (AA). Both EPA and LA bind in the active site with orientations similar to those seen previously with AA and dihomo-gamma-linolenic acid (DHLA). For EPA, the presence of an additional double bond (C-17/C-18) causes this substrate to bind in a "strained" conformation in which C-13 is misaligned with respect to Tyr-385, the residue that abstracts hydrogen from substrate fatty acids. Presumably, this misalignment is responsible for the low rate of EPA oxygenation. For LA, the carboxyl half binds in a more extended configuration than AA, which results in positioning C-11 next to Tyr-385. Val-349 and Ser-530, recently identified as important determinants for efficient oxygenation of DHLA by PGHS-1, play similar roles in the oxygenation of EPA and LA. Approximately 750- and 175-fold reductions in the oxygenation efficiency of EPA and LA were observed with V349A oPGHS-1, compared with a 2-fold change for AA. Val-349 contacts C-2 and C-3 of EPA and C-4 of LA orienting the carboxyl halves of these substrates so that the omega-ends are aligned properly for hydrogen abstraction. An S530T substitution decreases the V(max)/K(m) of EPA and LA by 375- and 140-fold. Ser-530 makes six contacts with EPA and four with LA involving C-8 through C-16; these interactions influence the alignment of the substrate for hydrogen abstraction. Interestingly, replacement of Phe-205 increases the volume of the cyclooxygenase site allowing EPA to be oxygenated more efficiently than with native oPGHS-1.

The epileptic process as nonlinear deterministic dynamics in a stochastic environment: an evaluation on mesial temporal lobe epilepsy.

The theory of deterministic chaos addresses simple deterministic dynamics in which nonlinearity gives rise to complex temporal behavior. Although biological neuronal networks such as the brain are highly complicated, a number of studies provide growing evidence that nonlinear time series analysis of brain electrical activity in patients with epilepsy is capable of providing potentially useful diagnostic information. In the present study, this analysis framework was extended by introducing a new measure xi, designed to discriminate between nonlinear deterministic and linear stochastic dynamics. For the evaluation of its discriminative power, xi was extracted from intracranial multi-channel EEGs recorded during the interictal state in 25 patients with unilateral mesial temporal lobe epilepsy. Strong indications of nonlinear determinism were found in recordings from within the epileptogenic zone, while EEG signals from other sites mainly resembled linear stochastic dynamics. In all investigated cases, this differentiation allowed to retrospectively determine the side of the epileptogenic zone in full agreement with results of the presurgical workup.

Mutational and X-ray crystallographic analysis of the interaction of dihomo-gamma -linolenic acid with prostaglandin endoperoxide H synthases.

Prostaglandin endoperoxide H synthases-1 and -2 (PGHSs) catalyze the committed step in prostaglandin biosynthesis. Both isozymes can oxygenate a variety of related polyunsaturated fatty acids. We report here the x-ray crystal structure of dihomo-gamma-linolenic acid (DHLA) in the cyclooxygenase site of PGHS-1 and the effects of active site substitutions on the oxygenation of DHLA, and we compare these results to those obtained previously with arachidonic acid (AA). DHLA is bound within the cyclooxygenase site in the same overall L-shaped conformation as AA. C-1 and C-11 through C-20 are in the same positions for both substrates, but the positions of C-2 through C-10 differ by up to 1.74 A. In general, substitutions of active site residues caused parallel changes in the oxygenation of both AA and DHLA. Two significant exceptions were Val-349 and Ser-530. A V349A substitution caused an 800-fold decrease in the V(max)/K(m) for DHLA but less than a 2-fold change with AA; kinetic evidence indicates that C-13 of DHLA is improperly positioned with respect to Tyr-385 in the V349A mutant thereby preventing efficient hydrogen abstraction. Val-349 contacts C-5 of DHLA and appears to serve as a structural bumper positioning the carboxyl half of DHLA, which, in turn, positions properly the omega-half of this substrate. A V349A substitution in PGHS-2 has similar, minor effects on the rates of oxygenation of AA and DHLA. Thus, Val-349 is a major determinant of substrate specificity for PGHS-1 but not for PGHS-2. Ser-530 also influences the substrate specificity of PGHS-1; an S530T substitution causes 40- and 750-fold decreases in oxygenation efficiencies for AA and DHLA, respectively.

Prostaglandin endoperoxide H synthase-1: the functions of cyclooxygenase active site residues in the binding, positioning, and oxygenation of arachidonic acid.

Prostaglandin endoperoxide H synthases (PGHSs) catalyze the committed step in the biosynthesis of prostaglandins and thromboxane, the conversion of arachidonic acid, two molecules of O(2), and two electrons to prostaglandin endoperoxide H(2) (PGH(2)). Formation of PGH(2) involves an initial oxygenation of arachidonate to yield PGG(2) catalyzed by the cyclooxygenase activity of the enzyme and then a reduction of the 15-hydroperoxyl group of PGG(2) to form PGH(2) catalyzed by the peroxidase activity. The cyclooxygenase active site is a hydrophobic channel that protrudes from the membrane binding domain into the core of the globular domain of PGHS. In the crystal structure of Co(3+)-heme ovine PGHS-1 complexed with arachidonic acid, 19 cyclooxygenase active site residues are predicted to make a total of 50 contacts with the substrate (Malkowski, M. G, Ginell, S., Smith, W. L., and Garavito, R. M. (2000) Science 289, 1933-1937); two of these are hydrophilic, and 48 involve hydrophobic interactions. We performed mutational analyses to determine the roles of 14 of these residues and 4 other closely neighboring residues in arachidonate binding and oxygenation. Mutants were analyzed for peroxidase and cyclooxygenase activity, and the products formed by various mutants were characterized. Overall, the results indicate that cyclooxygenase active site residues of PGHS-1 fall into five functional categories as follows: (a) residues directly involved in hydrogen abstraction from C-13 of arachidonate (Tyr-385); (b) residues essential for positioning C-13 of arachidonate for hydrogen abstraction (Gly-533 and Tyr-348); (c) residues critical for high affinity arachidonate binding (Arg-120); (d) residues critical for positioning arachidonate in a conformation so that when hydrogen abstraction does occur the molecule is optimally arranged to yield PGG(2) versus monohydroperoxy acid products (Val-349, Trp-387, and Leu-534); and (e) all other active site residues, which individually make less but measurable contributions to optimal catalytic efficiency.

The role of arginine 120 of human prostaglandin endoperoxide H synthase-2 in the interaction with fatty acid substrates and inhibitors.

Arg-120 is located near the mouth of the hydrophobic channel that forms the cyclooxygenase active site of prostaglandin endoperoxide H synthases (PGHSs)-1 and -2. Replacement of Arg-120 of ovine PGHS-1 with a glutamine increases the apparent Km of PGHS-1 for arachidonate by 1,000-fold (Bhattacharyya, D. K., Lecomte, M., Rieke, C. J., Garavito, R. M., and Smith, W. L. (1996) J. Biol. Chem. 271, 2179-2184). This and other evidence indicate that the guanido group of Arg-120 forms an ionic bond with the carboxylate group of arachidonate and that this interaction is an important contributor to the overall strength of arachidonate binding to PGHS-1. In contrast, we report here that R120Q human PGHS-2 (hPGHS-2) and native hPGHS-2 have very similar kinetic properties, but R120L hPGHS-2 catalyzes the oxygenation of arachidonate inefficiently. Our data indicate that the guanido group of Arg-120 of hPGHS-2 interacts with arachidonate through a hydrogen bond rather than an ionic bond and that this interaction is much less important for arachidonate binding to PGHS-2 than to PGHS-1. The Km values of PGHS-1 and -2 for arachidonate are the same, and all but one of the core residues of the active sites of the two isozymes are identical. Thus, the results of our studies of Arg-120 of PGHS-1 and -2 imply that interactions involved in the binding of arachidonate to PGHS-1 and -2 are quite different and that residues within the hydrophobic cyclooxygenase channel must contribute more significantly to arachidonate binding to PGHS-2 than to PGHS-1. As observed previously with R120Q PGHS-1, flurbiprofen was an ineffective inhibitor of R120Q hPGHS-2. PGHS-2-specific inhibitors including NS398, DuP-697, and SC58125 had IC50 values for the R120Q mutant that were up to 1,000-fold less than those observed for native hPGHS-2; thus, the positively charged guanido group of Arg-120 interferes with the binding of these compounds. NS398 did not cause time-dependent inhibition of R120Q hPGHS-2, whereas DuP-697 and SC58125 were time-dependent inhibitors. Thus, Arg-120 is important for the time-dependent inhibition of hPGHS-2 by NS398 but not by DuP-697 or SC58125.

Involvement of arginine 120, glutamate 524, and tyrosine 355 in the binding of arachidonate and 2-phenylpropionic acid inhibitors to the cyclooxygenase active site of ovine prostaglandin endoperoxide H synthase-1.

Examination of the crystal structure of the ovine prostaglandin endoperoxide synthase-1 (PGHS-1)/S- flurbiprofen complex (Picot, D., Loll, P.J., and Garavito, R.M. (1994) Nature 367, 243-2491) suggests (a) that the carboxyl group of arachidonic acid interacts with the arginino group of Arg120; (b) that Arg120 forms an important salt bridge with Glu524; and (c) that Tyr355, which is in close proximity to Arg120, could determine the stereochemical specificity of PGHS-1 toward 2-phenylpropionic acid inhibitors. To test these concepts, we used site-directed mutagenesis to prepare ovine PGHS-1 mutants having modifications of Arg120 (R120K, R120Q, R120E), Glu524 (E524D, E524Q, E524K), and Tyr355 (Y355F) and examined the properties of the mutant enzymes expressed in COS-1 cells. All of the mutants retained at least part of their cyclooxygenase and peroxidase activities except the R120E mutant, which had no detectable activity. The Km values of the R120K and R120Q mutants with arachidonic acid were 87 and 3300 microM, respectively, versus 4 microM for native PGHS-1. The R120Q mutant failed to undergo suicide inactivation during catalysis or time-dependent inhibition by flurbiprofen. These results are consistent with Arg120 binding the carboxylate group of arachidonate and suggest that interaction of the carboxylate group of substrates and inhibitors with Arg120 is necessary for suicide inactivation and time-dependent inhibition, respectively. The Km values for the E524D, E524Q, and E524K mutants were not significantly different from values obtained for the native PGHS-1, suggesting that this residue is not importantly involved in catalysis or substrate binding. The effect of modifications of Arg120 and Tyr355 on the stereospecificity of inhibitor binding was determined. Ratios of IC50 values for cyclooxygenase inhibition by D- and L-ibuprofen, a competitive cyclooxygenase inhibitor, were 32, 67, and 7.1 for native PGHS-1, R120Q PGHS-1, and Y355F PGHS-1, respectively. The decreased stereochemical specificity observed with the Y355F PGHS-1 mutant suggests that Tyr355 is a determinant of the stereospecificity of PGHS-1 toward inhibitors of the 2-phenylpropionic acid class.