Inhibition of voltage-dependent sodium channels by the anticonvulsant gamma-aminobutyric acid type A receptor modulator, 3-benzyl-3-ethyl-2-piperidinone

3-Benzyl-3-ethyl-2-piperidinone (3-BEP) belongs to a family of compounds that includes alpha- substituted gamma-butyrolactones, gamma-thiobutyrolactones, 2-pyrrolidinones and hexahydro-2H-azepin-2-ones. Many of these drugs exhibit potent in vivo anticonvulsant activity in mice. Previous electrophysiological studies demonstrated that they potentiate gamma-aminobutyric acid- (GABA) mediated chloride currents. This GABAA receptor modulation was thought to be the main mechanism of anticonvulsant activity. We report that 3-BEP also modulates sodium channels. It decreased sodium currents in cultured rat hippocampal neurons in a voltage- and concentration-dependent manner. The drug's apparent affinity increased as neurons were depolarized. At a holding potential of -60 mV, the apparent IC50 was 487 microM. This concentration is comparable to its EC50 for GABAA modulation (575 microM). Current blockade occurred over all activation voltages tested. The steady state inactivation curve was shifted by 600 microM 3-BEP from V50 = -65.3 mV to -72.0 mV, and recovery from inactivation was slowed from tau = 4.9 to 12.8 msec. Sodium current inhibition was not observed for three related compounds, suggesting a degree of chemical specificity for this activity. We conclude that in addition to its known effects on GABAA receptors, 3-BEP modulates sodium channels. Therefore this compound may prevent seizures by both enhancing inhibition and diminishing neuronal excitability.     

Evolution of the cytochrome P450 superfamily: sequence alignments and pharmacogenetics

The evolution of the cytochrome P450 (CYP) superfamily is described, with particular reference to major events in the development of biological forms during geological time. It is noted that the currently accepted timescale for the elaboration of the P450 phylogenetic tree exhibits close parallels with the evolution of terrestrial biota. Indeed, the present human P450 complement of xenobiotic-metabolizing enzymes may have originated from coevolutionary 'warfare' between plants and animals during the Devonian period about 400 million years ago. A number of key correspondences between the evolution of P450 system and the course of biological development over time, point to a mechanistic molecular biology of evolution which is consistent with a steady increase in atmospheric oxygenation beginning over 2000 million years ago, whereas dietary changes during more recent geological time may provide one possible explanation for certain species differences in metabolism. Alignment between P450 protein sequences within the same family or subfamily, together with across-family comparisons, aid the rationalization of drug metabolism specificities for different P450 isoforms, and can assist in an understanding of genetic polymorphisms in P450-mediated oxidations at the molecular level. Moreover, the variation in P450 regulatory mechanisms and inducibilities between different mammalian species are likely to have important implications for current procedures of chemical safety evaluation, which rely on pure genetic strains of laboratory bred rodents for the testing of compounds destined for human exposure.     

Electrochemical and spectroscopic properties of the iron-sulfur flavoprotein from Methanosarcina thermophila

An iron-sulfur flavoprotein (Isf) from the methanoarchaeaon Methanosarcina thermophila, which participates in electron transfer reactions required for the fermentation of acetate to methane, was characterized by electrochemistry and EPR and M?ssbauer spectroscopy. The midpoint potential (Em) of the FMN/FMNH2 couple was -0.277 V. No flavin semiquinone was observed during potentiometric titrations; however, low amounts of the radical were observed when Isf was quickly frozen after reaction with CO and the CO dehydrogenase/acetyl-CoA synthase complex from M. thermophila. Isf contained a [4Fe-4S]2+/1+ cluster with g values of 2.06 and 1.93 and an unusual split signal with g values at 1.86 and 1.82. The unusual morphology was attributed to microheterogeneity among Isf molecules. The Em value for the 2+/1+ redox couple of the cluster was -0.394 V. Extracts from H2-CO2-grown Methanobacterium thermoautotrophicum cells catalyzed either the H2- or CO-dependent reduction of M. thermophila Isf. In addition, Isf homologs were found in the genomic sequences of the CO2-reducing methanoarchaea M. thermoautotrophicum and Methanococcus jannaschii. These results support a general role for Isf in electron transfer reactions of both acetate-fermenting and CO2-reducing methanoarchaea. It is suggested that Isf functions to couple electron transfer from ferredoxin to membrane-bound electron carriers, such as methanophenazine and/or b-type cytochromes.     

The CYP2 family: models, mutants and interactions

1. The construction of three-dimensional models of mammalian cytochromes P450 from the CYP2 family is reported based on protein sequence alignment with CYP102, a bacterial P450 of known crystal structure. 2. The homology models of CYP2 family enzymes appear to show self-consistency with the currently accumulated information from site-directed mutagenesis and chemical modification of amino acid residues known to affect redox partner interactions. 3. The generation of these models from the recently reported crystal structure of substrate-bound CYP102 enables the exploration of likely active site contacts with specific substrates of CYP2 family isozymes.     

A high resolving power ion selector for post-source decay measurements in a reflecting time-of-flight mass spectrometer

An electrostatic deflector has been designed and constructed that can be used in a reflecting time-of-flight mass spectrometer for either single-deflector or dual-deflector velocity selection in post-source decay measurements. The deflector consists of an interleaved set of parallel deflection electrodes as in a Loeb/Cravath/Bradbury device, but thin metal ribbon instead of wire is used for the deflection electrodes. The time for reversing the electric field, which depends on various factors such as the electronics for pulsing the voltage and the time constant of a particular electrode geometry, is about 19 ns for the deflectors used in this study. By properly timing the reversal of the electric field, the time-window for ion transmission can be made substantially less than the switching time of each individual deflector. In conjunction with matrix-assisted laser desorption/ionization, the single-deflector's resolving power and transmission are robust with respect to laser fluence, i.e. they remain high even when the fluence is raised well above threshold. By contrast the operational features of the dual-deflector gate offer more versatility in locating and sizing the selection window. Operating the ion selector in a single-deflector mode, we have achieved a resolving power of approximately 710 full width at half maximum (FWHM) for different isotopes of protonated, sodiated, and potassiated substance-P (m/z 1348.6, 1370.6 and 1386.6 respectively; 10.073 keV). Operating it in the dual-deflector mode under two different sets of conditions, we have succeeded in obtaining resolving powers of approximately 1100 (FWHM) for protonated substance-P (m/z 1348.6; 10.8 keV) and approximately 5200 (FWHM) for an isotopomer of PEG 6000 (approximately m/z 6000; 10.04 keV). This accomplishment implies that high-resolution ion selection can be coupled to post-source decay analyses.     

Genomic variation in Trypanosoma cruzi clonal cultures

Spontaneous changes in restriction DNA profiles and pulsed-field gel electrophoresis (PFGE) patterns, along with a concomitant loss of infectivity, were observed in infective clones of Trypanosoma cruzi strain Y either following a number of passages during the exponential growth phase of after subcloning in liver infusion tryptone (LIT) medium using as the probe a genomic fragment of the parasite (pMYP16), indicating naturally occurring rearrangements of DNA sequences. No variation could be detected when the genomic DNA was probed with conserved T. cruzi tubulin and actin genes. There was no correlation between such rearrangements and the life-cycle forms of the parasites, since trypomastigote forms showed the same karyotype and hybridization patterns as did epimastigote forms. The variations observed could be reverted and infectivity, recovered after inoculation of the parasites in newborn mice.