Oxidation mechanism in the metabolism of (S)-N-[1-(3-morpholin-4-ylphenyl)ethyl]-3-phenylacrylamide on oxyferryl active site in CYP3A4 Cytochrome: DFT modeling

The metabolism mechanism of (S)-N-[1-(3-morpholin-4ylphenyl)ethyl]-3-phenylacrylamide, mediated by CYP3A4 Cytochrome has been investigated by density functional QM calculations aided with molecular mechanics/molecular dynamics simulations. Two different orientations of phenyl ring for substrate approach toward oxyferryl center, imposing two subsequent rearrangement pathways have been investigated. Starting from σ-complex in perpendicular orientation enzymatic mechanism involves consecutive proton shuttle intermediate, which further leads to the formation of alcohol and ketone. Parallel conformation leads solely to ketone product by 1,2 hydride shift. Although parallel and perpendicular σ-complexes are energetically equivalent both for the gas phase or PCM solvent model, molecular dynamics studies in full CYP3A4 environment show that perpendicular conformation of the σ-complex should be privileged, stabilized by hydrophobic interactions of phenylacrylamide chain. After assessing probability of the two conformations we postulate that the alcohol, accessible with the lowest energy barriers should be the major metabolite for studied substrate and CYP3A4 enzyme. Figure Orientation of phenyl ring towards porphyrin plane selected by substrate interaction with enzymatic cavity channels enzymatic reaction     

Heterologous expression of Pharaonis halorhodopsin in Xenopus laevis oocytes and electrophysiological characterization of its light-driven Cl- pump activity

Natronomonas pharaonis halorhodopsin (pHR) is an archaeal rhodopsin functioning as an inward-directed, light-driven Cl- pump. To characterize the electrophysiological features of the Cl- pump activity of pHR, we expressed pHR in Xenopus laevis oocytes and analyzed its photoinduced Cl- pump activity using the two-electrode voltage-clamp technique. Photoinduced outward currents were observed only in the presence of Cl-, Br-, I-, NO3-, and SCN-, but not in control oocytes, indicating that photoinduced anion currents were mediated by pHR. The relationship between photoinduced Cl- current via pHR and the light intensity was linear, demonstrating that transport of Cl- is driven by a single-photon reaction and that the steady-state current is proportional to the excited pHR molecule. The current-voltage relationship for pHR-mediated photoinduced currents was also linear between -150 mV and +50 mV. The slope of the line describing the current-voltage relationship increased as the number of the excited pHR molecules was increased by the light intensity. The reversal potential (VR) for Cl- as the substrate for the anion pump activity of pHR was about -400 mV. The value for VR was independent of light intensity, meaning that the VR reflects the intrinsic value of the excited pHR molecule. The value of VR changed significantly for the R123K mutant of pHR. We also show that the Cl- pump activity of pHR can generate a substantial negative membrane potential, indicating that pHR is a very potent Cl- pump. We have also analyzed the kinetics of voltage-dependent Cl- pump activity as well as that of the photocycle. Based on these data, a kinetic model for voltage-dependent Cl- transport via pHR is presented.     

Low frequency spectral density of ferrous heme: perturbations induced by axial ligation and protein insertion

Femtosecond coherence spectroscopy is used to probe low frequency (20-400 cm⁻¹) modes of the ferrous heme group in solution, with and without 2-methyl imidazole (2MeIm) as an axial ligand. The results are compared to heme proteins (CPO, P450_cam, HRP, Mb) where insertion of the heme into the protein results in redistribution of the low frequency spectral density and in (∼60%) longer damping times for the coherent signals. The major effect of imidazole ligation to the ferrous heme is the “softening” of the low frequency force constants by a factor of ∼0.6 ± 0.1. The functional consequences of imidazole ligation are assessed and it is found that the enthalpic CO rebinding barrier is increased significantly when imidazole is bound. The force constant softening analysis, combined with the kinetics results, indicates that the iron is displaced by only ∼0.2 Å from the heme plane in the absence of the imidazole ligand, whereas it is displaced by ∼0.4 Å when imidazole (histidine) is present. This suggests that binding of imidazole (histidine) as an axial ligand, and the concomitant softening of the force constants, leads to an anharmonic distortion of the heme group that has significant functional consequences.     

Kinetics of the reaction by which natural vitamin E is regenerated by vitamin C

The rate constant and activation energy of the regeneration reaction of natural vitamin E by vitamin C were determined with a double-mixing stopped-flow spectrophotometer. The formation of vitamin C radical was observed in the absorption spectrum. The kinetic effect of methyl substitution on the aromatic ring of vitamin E radical indicates that partial charge-transfer plays a role in the reaction. Since a substantial deuterium kinetic isotope effect was not found, the tunneling effect may not play an important role under the present experimental conditions.     

Identification of proteins whose expression is up- or down-regulated in the mushroom bodies in the honeybee brain using proteomics

To identify protein(s) with different expression patterns in the mushroom bodies (MBs) in the honeybee brain, we compared the protein profiles of MBs and optic lobes (OLs) using proteomics. Two-dimensional gel electrophoresis revealed that five and three spots were selectively expressed in the MBs or OLs, respectively. Liquid chromatography tandem mass spectrometry analysis identified juvenile hormone diol kinase and glyceraldehyde-3-phosphate dehydrogenase as MB- and OL-selective proteins, respectively. In situ hybridization revealed that jhdk expression was upregulated in MB neuron subsets, whereas gapdh expression was downregulated, indicating that MBs have a distinct gene and protein expression profile in the honeybee brain.     

Dual-labeled telomere sensing probes for quantification of telomerase activity assay

The present study describes an empirically discovered phenomenon that might be useful for development of a sensitive and rapid methodology for quantification of telomerase activity assay with simple data acquisition and possibility for calculation of telomerase product in absolute units. The method is based on the design and application of two single-stranded telomere sensing probes consisting of dual-labeled 16-mer oligonucleotides (fluorescent Cy3/Cy3-labeled and non-fluorescent IowaBlack/BHQ-labeled) that can simultaneously hybridize on the primary product of the telomerase reaction.     

Isolation and characterization of arbuscules from roots of an increased-arbuscule-forming mutant of Lotus japonicus

BACKGROUND AND AIMS: Previous methods for isolation of arbuscules from mycorrhizal roots are time-consuming, complex and expensive. Therefore, a simple, rapid and inexpensive method for the isolation of metabolically active arbuscules from plant root of an increased-arbuscule-forming mutant of Lotus japonicus (Ljsym78-2) is described. METHODS: Roots of the L. japonicus mutant plants Ljsym78-2 colonized by Glomus sp. were separated from soil, washed with water, immersed in CaSO(4) before being cut into 5-mm pieces and homogenized with a Waring blender at 6000 rpm for 30 s. The arbuscules were purified by separation from plant tissues with a 50-mum nylon mesh, finally collecting on a 30-mum nylon mesh. Enzyme histochemical staining showed that the collected arbuscules had succinate dehydrogenase, alkaline phosphatase and acid phosphatase activities. KEY RESULTS AND CONCLUSIONS: The enzymic activity of the arbuscules was not affected after the isolation process. The establishment of this simple, rapid and inexpensive method for the isolation of metabolically active arbuscules will be useful to clarify the biochemical processes occurring in nutrient exchange at the arbuscular interface.     

Induction of cambial reactivation by localized heating in a deciduous hardwood hybrid poplar (Populus sieboldii x P. grandidentata)

BACKGROUND AND AIMS: The timing of cambial reactivation plays an important role in the control of both the quantity and the quality of wood. The effect of localized heating on cambial reactivation in the main stem of a deciduous hardwood hybrid poplar (Populus sieboldii x P. grandidentata) was investigated. METHODS: Electric heating tape (20-22 degrees C) was wrapped at one side of the main stem of cloned hybrid poplar trees at breast height in winter. Small blocks were collected from both heated and non-heated control portions of the stem for sequential observations of cambial activity and for studies of the localization of storage starch around the cambium from dormancy to reactivation by light microscopy. KEY RESULTS: Cell division in phloem began earlier than cambial reactivation in locally heated portions of stems. Moreover, the cambial reactivation induced by localized heating occurred earlier than natural cambial reactivation. In heated stems, well-developed secondary xylem was produced that had almost the same structure as the natural xylem. When cambial reactivation was induced by heating, the buds of trees had not yet burst, indicating that there was no close temporal relationship between bud burst and cambial reactivation. In heated stems, the amount of storage starch decreased near the cambium upon reactivation of the cambium. After cambial reactivation, storage starch disappeared completely. Storage starch appeared again, near the cambium, during xylem differentiation in heated stems. CONCLUSIONS: The results suggest that, in deciduous diffuse-porous hardwood poplar growing in a temperate zone, the temperature in the stem is a limiting factor for reactivation of phloem and cambium. An increase in temperature might induce the conversion of storage starch to sucrose for the activation of cambial cell division and secondary xylem. Localized heating in poplar stems provides a useful experimental system for studies of cambial biology.