Subjective sleep quality in premenstrual syndrome

Premenstrual syndrome (PMS) is a cyclical disorder observed in late luteal phase and presenting with behavioral changes that can affect interpersonal relationships and normal daily activity. Sleep disturbances are also common. The aim of this study is to investigate the relationship between PMS and subjective sleep quality with Pitsburg Sleep Quality Index (PSQI) in the Medical Academy students, whom have considerable information about menstruation. PMS was detected with "Premenstrual Syndrome Scale", and PSQI was used to evaluate subjective sleep quality. Chi-square test and Kendall's rank correlation analysis were used in statistical analysis. p values (p < 0.05) were considered as statistical significant. Poor sleep quality was found in the 75.6% of the participants with PMS, and 58.8% of the participants without PMS (p < 0.05). Only component 5 (sleep disorder component) of the PSQI components revealed statistically significant difference (1.7 ± 0.6 in participants with PMS, and 1.5 ± 0.6 without PMS, p < 0.05). There was a positive correlation between total PSQI score and all of its' components, except component 6 (sleeping pill usage component) (p < 0.05). The strongest association was found to be in the component 5 (r = 0.528; p = 0.0001). Results of our study suggested the poor sleep quality due to sleep disorders in women with PMS.     

Cooperativity in oxidation reactions catalyzed by cytochrome P450 1A2: highly cooperative pyrene hydroxylation and multiphasic kinetics of ligand binding

Rabbit liver cytochrome P450 (P450) 1A2 was found to catalyze the 5,6-epoxidation of alpha-naphthoflavone (alphaNF), 1-hydroxylation of pyrene, and the subsequent 6-, 8-, and other hydroxylations of 1-hydroxy (OH) pyrene. Plots of steady-state rates of product formation versus substrate concentration were hyperbolic for alphaNF epoxidation but highly cooperative (Hill n coefficients of 2-4) for pyrene and 1-OH pyrene hydroxylation. When any of the three substrates (alphaNF, pyrene, 1-OH pyrene) were mixed with ferric P450 1A2 using stopped-flow methods, the changes in the heme Soret spectra were relatively slow and multiphasic. Changes in the fluorescence of all of the substrates were much faster, consistent with rapid initial binding to P450 1A2 in a manner that does not change the heme spectrum. For binding of pyrene to ferrous P450 1A2, the course of the spectra revealed sequential changes in opposite directions, consistent with P450 1A2 being involved in a series of transitions to explain the kinetic multiphasicity as opposed to multiple, slowly interconverting populations of enzyme undergoing the same event at different rates. Models of rabbit P450 1A2 based on a published crystal structure of a human P450 1A2-alphaNF complex show active site space for only one alphaNF or for two pyrenes. The spectral changes observed for binding and hydroxylation of pyrene and 1-OH pyrene could be fit to a kinetic model in which hydroxylation occurs only when two substrates are bound. Elements of this mechanism may be relevant to other cases of P450 cooperativity.     

Cooperativity of cytochrome P450 1A2: interactions of 1,4-phenylene diisocyanide and 1-isopropoxy-4-nitrobenzene

Homotropic cooperativity of 1-alkoxy-4-nitrobenzene substrates and also their heterotropic cooperative binding interactions with the iron ligand 1,4-phenylene diisocyanide (Ph(NC)₂) had been demonstrated previously with rabbit cytochrome P450 (P450) 1A2 [G.P. Miller, F.P. Guengerich, Biochemistry 40 (2001) 7262-7272]. Multiphasic kinetics were observed for the binding of Ph(NC)₂ to both ferric and ferrous P450 1A2, including relatively slow steps. Ph(NC)₂ induced an apparently rapid change in the circular dichroism spectrum, consistent with a structural change, but had no effect on tryptophan fluorescence. Ph(NC)₂ binds the P450 iron in both the ferric and ferrous forms; ferric P450 1A2 was reduced rapidly in the absence of added ligands, and the rate was attenuated when Ph(NC)₂ was bound. No oxidation products of Ph(NC)₂ were detected. Docking studies with a rabbit P450 1A2 homology model based on the published structure of a human P450 1A2·α-naphthoflavone (αNF) complex indicated adequate room for a complex with either two 1-isopropoxy-4-nitrobenzene molecules or a combination of one 1-isopropoxy-4-nitrobenzene and one Ph(NC)₂; in the case of αNF no space for an extra ligand was available. The patterns of homotropic cooperativity seen with 1-alkoxy-4-nitrobenzenes (biphasic plots of v vs. S) differ from those seen with polycyclic hydrocarbons (positive cooperativity), suggesting that only with the latter does the ligand interaction produce improved catalysis. Consistent with this view, Ph(NC)₂ inhibited the oxidation of 1-isopropoxy-4-nitrobenzene and other substrates.     

Functional motions of influenza virus hemagglutinin: a structure-based analytical approach.

Influenza virus hemagglutinin (HA), a homotrimeric integral membrane glycoprotein essential for viral infection, is engaged in two biological functions: recognition of target cells' receptor proteins and fusion of viral and endosomal membranes, both requiring substantial conformational flexibility from the part of the glycoprotein. The different modes of collective motions underlying the functional mobility/adaptability of the protein are determined in the present study using an extension of the Gaussian network model (GNM) to treat concerted anisotropic motions. We determine the molecular mechanisms that may underlie HA function, along with the structural regions or residues whose mutations are expected to impede function. Good agreement between theoretically predicted fluctuations of individual residues and corresponding x-ray crystallographic temperature factors is found, which lends support to the GNM elucidation of the conformational dynamics of HA by focusing upon a subset of dominant modes. The lowest frequency mode indicates a global torsion of the HA trimer about its longitudinal axis, accompanied by a substantial mobility at the viral membrane connection. This mode is proposed to constitute the dominant molecular mechanism for the translocation and aggregation of HAs, and for the opening and dilation of the fusion pore. The second and third collective modes indicate a global bending, allowing for a large lateral surface exposure, which is likely to facilitate the close association of the viral and endosomal membranes before pore opening. The analysis of kinetically hot residues, in contrast, reveals a localization of energy centered around the HA2 residue Asp112, which apparently triggers the solvent exposure of the fusion peptide.     

Complex reactions catalyzed by cytochrome P450 enzymes

Cytochrome P450 (P450) enzymes are some of the most versatile redox proteins known. The basic P450 reactions include C-hydroxylation, heteroatom oxygenation, heteroatom release (dealkylation), and epoxide formation. Mechanistic explanations for these reactions have been advanced. A number of more complex P450 reactions also occur, and these can be understood largely in the context of the basic chemical mechanisms and subsequent rearrangements. The list discussed here updates a 2001 review and includes chlorine oxygenation, aromatic dehalogenation, formation of diindole products, dimer formation via Diels-Alder reactions of products, ring coupling and also ring formation, reductive activation (e.g., aristolochic acid), ring contraction (piperidine nitroxide radical), oxidation of troglitazone, cleavage of amino oxazoles and a 1,2,4-oxadiazole ring, bioactivation of a dihydrobenzoxathiin, and oxidative aryl migration.     

Extraction and Inhibition of Enzymatic Activity of Botulinum Neurotoxins/A1, /A2, and /A3 by a Panel of Monoclonal Anti-BoNT/A Antibodies

Botulinum neurotoxins (BoNTs) are extremely potent toxins that are capable of causing death or respiratory failure leading to long-term intensive care. Treatment includes serotype-specific antitoxins, which must be administered early in the course of the intoxication. Rapidly determining human exposure to BoNT is an important public health goal. In previous work, our laboratory focused on developing Endopep-MS, a mass spectrometry-based endopeptidase method for detecting and differentiating BoNT/A–G serotypes in buffer and BoNT/A, /B, /E, and /F in clinical samples. We have previously reported the effectiveness of antibody-capture to purify and concentrate BoNTs from complex matrices, such as clinical samples. Because some antibodies inhibit or neutralize the activity of BoNT, the choice of antibody with which to extract the toxin is critical. In this work, we evaluated a panel of 16 anti-BoNT/A monoclonal antibodies (mAbs) for their ability to inhibit the in vitro activity of BoNT/A1, /A2, and /A3 complex as well as the recombinant LC of A1. We also evaluated the same antibody panel for the ability to extract BoNT/A1, /A2, and /A3. Among the mAbs, there were significant differences in extraction efficiency, ability to extract BoNT/A subtypes, and inhibitory effect on BoNT catalytic activity. The mAbs binding the C-terminal portion of the BoNT/A heavy chain had optimal properties for use in the Endopep-MS assay.     

Computational study of the intramolecular proton transfer reactions of 3-hydroxytropolone (2,7-dihydroxycyclohepta-2,4,6-trien-1-one) and its dimers

The proton transfer reaction and dimerization processes of 3-hydroxytropolone (3-OHTRN) have been investigated using density functional theory (DFT) at the B3LYP/6–31+G** level. The influence of the solvent on the proton transfer reaction of 3-OHTRN was examined using the self-consistent isodensity polarized continuum model (SCI-PCM) with different dielectric constants (ε?=?4.9, CHCI₃; ε?=?32.63, CH₃OH; ε?=?78.39, H₂O). The intramolecular proton transfer reaction occurs more readily in the gas phase than in solution. Results also show that the stability of 3-OHTRN dimers in the gas phase is directly affected by the hydrogen bond length in the dimer structure.     

Kinetic deuterium isotope effects for 7-alkoxycoumarin O-dealkylation reactions catalyzed by human cytochromes P450 and in liver microsomes. Rate-limiting C-H bond breaking in cytochrome P450 1A2 substrate oxidation

7-Ethoxy (OEt) coumarin has been used as a model substrate in many cytochrome P450 (P450) studies, including the use of kinetic isotope effects to probe facets of P450 kinetics. P450s 1A2 and 2E1 are known to be the major catalysts of 7-OEt coumarin O-deethylation in human liver microsomes. Human P450 1A2 also catalyzed 3-hydroxylation of 7-methoxy (OMe) coumarin at appreciable rates but P450 2E1 did not. Intramolecular kinetic isotope effects were used as estimates of the intrinsic kinetic deuterium isotope effects for both 7-OMe and 7-OEt coumarin dealkylation reactions. The apparent intrinsic isotope effect for P450 1A2 (9.4 for O-demethylation, 6.1 for O-deethylation) showed little attenuation in other competitive and noncompetitive experiments. With P450 2E1, the intrinsic isotope effect (9.6 for O-demethylation, 6.1 for O-deethylation) was attenuated in the noncompetitive intermolecular experiments. High noncompetitive intermolecular kinetic isotope effects were seen for 7-OEt coumarin O-deethylation in a baculovirus-based microsomal system and five samples of human liver microsomes (7.3-8.1 for O-deethylation), consistent with the view that P450 1A2 is the most efficient P450 catalyzing this reaction in human liver microsomes and indicating that the C-H bond-breaking step makes a major contribution to the rate of this P450 (1A2) reaction. Thus, the rate-limiting step appears to be the chemistry of the breaking of this bond by the activated iron-oxygen complex, as opposed to steps involved in the generation of the reactive complex. The conclusion about the rate-limiting step applies to all of the systems studied with this model P450 1A2 reaction including human liver microsomes, the most physiologically relevant.     

Muscular dystrophies: genes to pathogenesis

Muscular dystrophies are a genetically heterogeneous group of degenerative muscle disorders. Nearly 30 genes are known to give rise to various forms of muscular dystrophy, which differ in age of onset, severity, and muscle groups affected. The number of genes identified increases each year, adding to our understanding as well as revealing the overall complexity of the pathogenesis of these diseases.     

A novel and selective monoamine oxidase B substrate

Cyclic five- and six-membered tertiary allylamines constitute a unique class of monoamine oxidase substrates that undergo a net two-electron alpha-carbon oxidation to form the cyclic, conjugated eniminium metabolites. The corresponding saturated pyrrolidinyl and piperidinyl systems are not substrates for this flavoenzyme system. In an attempt to evaluate possible contributions that pi-orbital stabilization of the putative alpha-carbon radical intermediates may play in the catalytic pathway, we have examined the substrate properties of 3-methyl-6-phenyl-3-aza-bicyclo[4.1.0]heptane, the 3,4-cyclopropyl analog of the selective monoamine oxidase B substrate 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP). The results, which document the first reported example of a saturated, cyclic tertiary amine with monoamine oxidase substrate properties, are consistent with alpha-carbon radical stabilization as a contributing factor in the catalytic pathway.