Unveiling controlling factors of the S0/S1 minimum-energy conical intersection (3): Frozen orbital analysis based on the spin-flip theory.

Conical intersections (CIs), which indicate the crossing of two or more adiabatic electronic states, are crucial in the mechanisms of photophysical, photochemical, and photobiological processes. Although various geometries and energy levels have been reported using quantum chemical calculations, the systematic interpretation of the minimum energy CI (MECI) geometries is unclear. A previous study [Nakai et al., J. Phys. Chem. A 122, 8905 (2018)] performed frozen orbital analysis (FZOA) based on time-dependent density functional theory (TDDFT) at the MECI formed between the ground and first electronic excited states (S0/S1 MECI), thereby inductively clarifying two controlling factors. However, one of the factors that the highest occupied molecular orbital (HOMO) and the lowest unoccupied molecular orbital (LUMO) energy gap became close to the HOMO-LUMO Coulomb integral was not valid in the case of spin-flip TDDFT (SF-TDDFT), which is frequently used as a means of the geometry optimization of MECI [Inamori et al., J. Chem. Phys. 152, 144108 (2020)]. This study revisited the controlling factors using FZOA for the SF-TDDFT method. Based on spin-adopted configurations within a minimum active space, the S0-S1 excitation energy is approximately represented by the HOMO and LUMO energy gap ΔεHL, a contribution from Coulomb integrals JHL″ and that from the HOMO-LUMO exchange integral KHL″. Furthermore, numerical applications of the revised formula at the SF-TDDFT method confirmed the control factors of S0/S1 MECI.

Changes in beta and high-gamma power in resting-state electrocorticogram induced by repetitive transcranial magnetic stimulation of primary motor cortex in unanesthetized macaque monkeys.

Repetitive transcranial magnetic stimulation (rTMS) is now widely used as a means of neuromodulation, but the details of the mechanisms by which rTMS works remain unclarified. As a step forward to unveiling the neural phenomena occurring underneath the TMS coil, we conducted an electrophysiological study using awake and unanesthetized monkeys with subdural electrocorticogram (ECoG) electrodes implanted over the primary motor cortex (MI). We evaluated the effects of low-frequency (1 Hz) and high-frequency (10 Hz) rTMS on the resting-state ECoG signals in the stimulated MI, as well as the motor evoked potentials (MEPs) in the contralateral hand. Following the 1-Hz rTMS application, the ECoG beta band power and the MEP amplitude were significantly decreased. Following the 10-Hz rTMS application, the ECoG high-gamma power and the MEP amplitude significantly increased. Given that beta and high-gamma activities in the ECoG reflect the synchronous firing and the firing frequency of cell assemblies, respectively, in local neural circuits, these results suggest that low-frequency rTMS inhibits neural activity by desynchronizing the firing activity of local circuits, whereas high-frequency rTMS facilitates neural activity by increasing the firing rate of cell assemblies in the local circuits.

Purification and characterization of cathepsin B-like cysteine protease from cotyledons of daikon radish, Raphanus sativus.

Plant cathepsin B-like cysteine protease (CBCP) plays a role in disease resistance and in protein remobilization during germination. The ability of animal cathepsin B to function as a dipeptidyl carboxypeptidase has been attributed to the presence of a dihistidine (His110-His111) motif in the occluding loop, which represents a unique structure of cathepsin B. However, a dihistidine motif is not present in the predicted sequence of the occluding loop of plant CBCP, as determined from cDNA sequence analysis, and the loop is shorter. In an effort to investigate the enzymatic properties of plant CBCP, which possesses the unusual occluding loop, we have purified CBCP from the cotyledons of daikon radish (Raphanus sativus) by chromatography through Sephacryl S-200, DEAE-cellulose, hydroxyapatite and organomercurial-Sepharose. The molecular mass of the enzyme was estimated to be 28 kDa by SDS/PAGE under reducing conditions. The best synthetic substrate for CBCP was t-butyloxycarbonyl Leu-Arg-Arg-4-methylcoumaryl 7-amide, as is the case with human cathepsin B. However, the endopeptidase activity of CBCP towards glucagon and adrenocorticotropic hormone showed broad cleavage specificity. Human cathepsin B preferentially cleaves model peptides via its dipeptidyl carboxypeptidase activity, whereas daikon CBCP displays both endopeptidase and exopeptidase activities. In addition, CBCP was found to display carboxymonopeptidase activity against the substrate o-aminobenzoyl-Phe-Arg-Phe(4-NO(2)). Daikon CBCP is less sensitive (1/7000) to CA-074 than human cathepsin B. Expression analysis of CBCP at the protein and RNA levels indicated that daikon CBCP activity in cotyledons is regulated by post-transcriptional events during germination.