Hiroshi Nakajima

Effects of extracts and neferine from the embryo of Nelumbo nucifera seeds on the central nervous system

The effects of embryos of the seeds of Nelumbo nucifera on the central nervous system were studied in mice. MeOH extracts of embryos of Nelumbo nucifera seeds significantly inhibited locomotor activity in mice. The MeOH extract was successively partitioned between H(2)O and n-hexane, between H(2)O and CHCl(3), and between H(2)O and n-BuOH. CHCl(3) extracts strongly inhibited locomotor activity in mice, although other extracts had no effect on locomotor activity. The main alkaloid of CHCl(3) extracts, neferine, dose-dependently inhibited locomotor activity in mice. Neferine induced hypothermia in mice and apparently potentiated thiopental-induced sleeping time. An anxiolytic, diazepam, decreased locomotor activity, rectal temperature and enhanced sleep elicited by thiopental, similar to neferine. In addition, neferine and diazepam showed anti-anxiety effects in the elevated plus maze test. Neferine did not affect muscle coordination by the rota-rod test. Neferine did not affect strychnine- nor picrotoxin-induced seizure. In contrast, diazepam had apparent muscle relaxant and anti-convulsant effects. These results suggest that neferine has several central effects and that neferine may participate in the efficacy of the sedative effects of embryos of the seeds of Nelumbo nucifera. The mechanisms of the sedative effects of neferine are not similar to those of diazepam.

Intercalation and polymerization of 4-anilinoaniline and 4-anilinoanilinium iodide in the VOPO4 and V2O5 interlayer spaces

Reactions of 4-anilinoaniline and 4-anilinoanilinium iodide with powdered VOPO4·2H2O suspended in ethanol gave compounds intercalated with the 4-anilinoanilinium cation. Reactions of 4-anilinoaniline and its hydrogen iodide salt with powdered V2O5·nH2O suspended in ethanol resulted in the direct oxidative polymerization of 4-anilinoanilinium moieties in the V2O5 Interlayer space. The polymerization processes of 4-anilinoaniline and its hydrogen iodide salt in the VOPO4 and V2O5 Interlayer spaces are discussed on the basis of powder X-ray diffraction patterns, infrared and X-ray photoelectron spectroscopies as well as gel-permeation chromatography.

Antidepressant-like effects of neferine in the forced swimming test involve the serotonin1A (5-HT1A) receptor in mice.

The effects of neferine, an alkaloid of Nelumbo nucifera Gaertner embryos, on immobility in the forced swimming test, which is used to evaluate antidepressants, were investigated in mice. The administration of neferine from 25 to 100 mg/kg i.p. elicited anti-immobility effects in mice. The molecular dose effects of neferine in the forced swimming test were almost equal to those of the typical antidepressants maprotiline and imipramine. The involvement of the 5-HT receptor subtypes was also studied using 5-HT receptor antagonists. Anti-immobility effects of neferine are antagonized by the serotonin1A (5-HT1A) receptor antagonist, N-[2-[4-(2-methoxyphenyl)-1-piperazinyl]ethyl]-N-(2-pyridinyl)cyclohexanecarboxamide (WAY 100635). However, the 5-HT1B receptor antagonist, 3-[3-(dimethylamino)propyl]-4-hydroxy-N-[4-(4-pyridinyl)phenyl] benzamide dihydrochloride (GR 55562), the 5-HT2 receptor antagonist, 6-methyl-1-(methylethyl)-ergoline-8beta-carboxylic acid 2-hydroxy-1-methylpropyl ester (LY 53857), the 5-HT3 receptor antagonist, ondansetron and the 5-HT4 receptor antagonist, 4-amino-5-chloro-2-methoxy-benzoic acid 2-(diethylamino)ethyl ester (SDZ 205,557) did not affect the anti-immobility effects of neferine. The anti-immobility effect of the selective 5-HT1A receptor agonist, 8-hydroxy-2-(di-n-propylamino)tetaralin (8-OH-DPAT) was also antagonized by WAY 100635. Furthermore, co-administration of subactive doses of neferine (10 mg/kg) and 8-OH-DPAT (0.1 mg/kg) produced synergistic antidepressant-like effects. These results suggest that neferine shows antidepressant-like effects in mice similar to typical antidepressants and that these effects are mediated by the 5-HT1A receptor. Therefore, the central effects of neferine are likely to be linked to serotonergic neurotransmission.

Mechanism of Intramolecular Electron Transfer in the Photoexcited Zn-Substituted Cytochrome c : Theoretical and Experimental Perspective

Photoinduced electron transfer (ET) in zinc-substituted cytochrome c (Zn-cyt c) has been utilized in many studies on the long-range ET in protein. Attempting to understand its ET mechanism in terms of electronic structure of the molecule, we have calculated an all-electron wave function for the ground-state of Zn-cyt c on the basis of density functional theory (DFT). The four molecular orbitals (MOs) responsible for excitation by UV-vis light (Goutermans 4-orbitals) are assigned on the basis of the excited states of chromophore model for Zn-porphine complex calculated with the time-dependent DFT method. ET rates between each Goutermans 4-orbitals and other MOs were estimated using Fermis golden rule. It appeared that the two occupied MOs of the 4-orbitals show exclusively higher ET rate from/to particular MOs that localize on outermost amino acid residues (Lys 7 or Asn 54), respectively, whereas ET rates involving the two unoccupied MOs of the 4-orbitals are much slower. These results imply that the intramolecular ET in photoexcited Zn-cyt c is governed by the hole transfer through occupied MOs. The couplings of MOs between zinc porphyrin core and specific amino acid residues on the protein surface have been demonstrated in Zn-cyt c immobilized on an Au electrode via carboxylic acid group-terminated self-assembled monolayer. The Zn-cyt c-modified electrode showed photocurrents responsible for photoillumination. The action spectrum of the photocurrent was identical with the absorption spectrum of Zn-cyt c, indicating photoinduced electron conduction via occupied MOs. The voltage dependence of the photocurrent appeared to be linear and bidirectional like a photoconductor, which strongly supports the intramolecular ET mechanism in Zn-cyt c proposed on the basis of the theoretical calculations.

Characterization of peroxide-bound heme species generated in the reaction of thermally tolerant cytochrome c552 with hydrogen peroxide.

Peroxide‐bound heme species have been considered difficult to detect under physiological conditions because of their intrinsically transient properties. Cytochrome c552 (cyt c552), from Thermus thermophirus HB8, bearing a mutation to an alanine at Met69 (M69A) reacts with hydrogen peroxide (H2O2) to generate a detectable hydroperoxo‐ferric heme ([Fe3+uf8ffOOH]) species at ambient temperature. EPR measurements during appropriate reaction periods reveal that the [Fe3+uf8ffOOH] species is in a preequilibrium state between the resting form of the cyt c552 variant and a subsequent intermediate, compound II with a protein radical. Addition of ascorbic acid to the reaction mixture of the cyt c552 variant and H2O2 does not affect the formation of the [Fe3+uf8ffOOH] species,a result suggesting that the species is incompetent for the oxidation of even an oxidatively fragile substrate such as ascorbic acid. Another variant bearing an additional mutation to aspartic acid at Val49 (V49D/M69A) reveals that a highly hydrophobic heme cavity in cyt c552 accounts for the generation of the durable [Fe3+uf8ffOOH] species. The less polar environment inside the cavity is expected to prevent H2O from approaching the cavity. This would suppress protonation of the distal oxygen atom of the [Fe3+uf8ffOOH] species and retard subsequent dissociation of H2O from the OOH moiety.

Engineering of Thermus thermophilus Cytochrome C552 : Thermally Tolerant Artificial Peroxidase

Application of peroxidases as catalysts for the oxidation of a wide spectrum of organic compounds has been a major focus in industrial sectors such as food processing, bioremediation, and biorefinement of oil because they use hydrogen peroxide (H2O2), an environmentally low-load oxidant. The full-scale use of peroxidases is, however, restricted by poor thermal and environmental stability, as is the case with many commercially available enzymes. Therefore, peroxidases with improved thermal stabilities are highly desired. Their detection in thermophilic bacteria by screening techniques is a popular approaches to finding thermally tolerant enzymes, and several such peroxidases have been isolated and characterized. 3] Although these peroxidases show remarkable stabilities and enzymatic activities at elevated temperatures in their host strains or cell lysates, the properties of the purified enzymes are still not sufficient for industrial applications. Some of the enzymes undergo rapid loss of activity in the presence of H2O2 over physiological levels. Partial compensation for the reduced thermoACHTUNGTRENNUNGstabilities and activities of the purified enzymes might be achievable by random mutagenesis achieved through evolutionary engineering. However, there are difficulties in elucidating the obtained variations in chemical terms, which prevents further tuning to improve the desired properties. Here we demonstrate that cytochrome c552 (Cyt c552) from Thermus thermophilus HB8 can be transformed into a thermally stable artificial peroxidase by rational modification based on the molecular mechanisms of natural peroxidases. Cyt c552 is an electron transfer protein containing His15 and Met69 as heme axial ligands, and it has characteristically high stability against thermal denaturation. In this study, two amino acid residues in Cyt c552 were chosen for mutagenesis in line with the peroxidase mechanism and three-dimensional structure of the protein (PDB ID: 1C52). One was Met69, which was replaced with alanine in order to provide a reaction site immediately above the heme iron (M69A). Another was the mutagenesis of Val49 to aspartic acid (V49D), which would be expected to introduce general acid–base catalyst, a fundamental element of the peroxidase mechanism, in the heme cavity. Although weak peroxidase activity is a natural property of cytochrome c, a variant bearing both mutations (M69A/V49D) exerts enhanced activity, which is observed neither for the wild-type nor for variants bearing only the M69A mutation. Figure 1 shows the temperature dependence of CD spectra recorded at 222 nm for the Cyt c552 variants (M69A/V49D and M69A) together with the wild-type and the H64D myoglobin variant (Mb H64D), an engineered sperm whale myoglobin

Protein Photoconductors and Photodiodes

Long-range electron transfer (ET) through proteins plays important roles in living systems. ET proteins can be regarded as wide-band-gap molecular semiconductors with “dopants” (redox or photoactive species) that provide an electronor hole-localizing site, though the protein scaffold has insulating character. Intramolecular ET in proteins has been investigated in detail by many groups to elucidate the factors that control the rates of these nonadiabatic reactions. 11–13] Dutton and co-workers proposed a square barrier ET rate model, while Gray and colleagues showed that ET depends on the structure of the medium between an electron donor and acceptor. We have investigated photoinduced ET in two proteins, zinc-substituted cytochrome b562 (Zn-cyt b562) and zinc-substituted cytochrome c (Zn-cyt c), the semiconductor properties of which depend on the charge distribution on their molecular surfaces: Zn-cyt b562 has n-type semiconductor character while Zn-cyt c is p-type, although the active center of the two systems is virtually identical. This finding may open up the world of protein-based electronics, because the semiconductor character of proteins could be controlled by variations in surface charge. Zn-cyt b562 on the H2N-SAM/Au electrode (SAM = selfassembled monolayer) was prepared according to the literature (see the Supporting Information). The orientation of Zn-cyt b562 on the H2N-SAM/Au surface (Figure 1) is optimal for favorable heme-region (acidic patch) electrostatic interactions with the SAM positive charge. Figure 2a shows cathodic photocurrents in response to switching on and off of 420 nm illumination for bias voltages of + 300, 0, and 300 mV (vs. Ag jAgCl). The action spectrum (Figure 2b) is similar to the absorption spectrum of Zn-cyt b562 (Figure 2b, inset), which suggests that the photocurrent originates in the protein. Incidentally, there is no photocurrent response in the case that protein changes Zncytb562 into Fe-cytb562. Notably, the Q-band (550 nm) photocurrent response is more prominent in comparison with the absorption spectrum, that is, the Soret/Q ratio in absorbance (I420/I550) of the action spectrum is 3.7 whereas that of the UV/ Vis spectrum is 11.1, thus implying that the photocurrent is derived from migration of the photoexcited electron. These findings are consistent with this mechanism: 1) the photocurrent was observed only in the cathodic direction under the range of bias potential employed here (Figure 2c), and 2) addition of methyl viologen (E0 = 0.62 V vs. Ag j AgCl) to the solution as a redox reagent enhanced the cathodic photocurrent, while addition of potassium ferri/ ferrocyanide (E0 =+ 0.17 V vs. Ag jAgCl) did not show such an effect. 18] This is quite in contrast to the Zn-cyt c/HOOC-SAM/Au system, where the photocurrent action spectrum can be superimposed on the UV/Vis spectrum and the addition of the ferri/ferrocyanide effectively increases the photocurrent that derives from the hole in the occupied molecular orbitals (MOs), or the valence band. Thus, although Zn-cyt b562 and Zn-cyt c are derivatives from similar ET proteins, the systems have a different electronic nature: a photodiode (n-type) and a photoconductor (p-type) character, respectively (Figure 2c). To elucidate the electronic structure and the factors that control the semiconductor properties of the two Zn-cytochrome proteins, we carried out all-electron DFT calculations for Zn-cyt b562. We estimated the photoinduced ET rate constants for Zn-cyt c [Eq. (1)]:

The role of the Fe-S cluster in the sensory domain of nitrogenase transcriptional activator VnfA from Azotobacter vinelandii.

Transcriptional activator VnfA is required for the expression of a second nitrogenase system encoded in the vnfH and vnfDGK operons in Azotobacter vinelandii. In the present study, we have purified full‐length VnfA produced in E. coli as recombinant proteins (Strep‐tag attached and tag‐less proteins), enabling detailed characterization of VnfA for the first time. The EPR spectra of whole cells producing tag‐less VnfA (VnfA) show distinctive signals assignable to a 3Fe‐4S cluster in the oxidized form ([Fe3S4]+). Although aerobically purified VnfA shows no vestiges of any Fe‐S clusters, enzymatic reconstitution under anaerobic conditions reproduced [Fe3S4]+ dominantly in the protein. Additional spectroscopic evidence of [Fe3S4]+inu2003vitro is provided by anaerobically purified Strep‐tag attached VnfA. Thus, spectroscopic studies both inu2003vivo and inu2003vitro indicate the involvement of [Fe3S4]+ as a prosthetic group in VnfA. Molecular mass analyses reveal that VnfA is a tetramer both in the presence and absence of the Fe‐S cluster. Quantitative data of iron and acid‐labile sulfur in reconstituted VnfA are fitted with four 3Fe‐4S clusters per a tetramer, suggesting that one subunit bears one cluster. Inu2003vivoβ‐gal assays reveal that the Fe‐S cluster which is presumably anchored in the GAF domain by the N‐terminal cysteine residues is essential for VnfA to exert its transcription activity on the target nitrogenase genes. Unlike the NifAL system of A. vinelandii, O2 shows no effect on the transcriptional activity of VnfA but reactive oxygen species is reactive to cause disassembly of the Fe‐S cluster and turns active VnfA inactive.

Azurin–Poly(N‐isopropylacrylamide) Conjugates by Site‐Directed Mutagenesis and their Thermosensitive Behavior in Electron‐Transfer Processes

Searching for intelligence: Azurin-PNIPAM conjugates were prepared by site-directed mutagenesis followed by protein reconstitution by using imidazole-conjugated poly(N-isopropylacrylamides). The polymer-bound imidazole acts as a ligand in the active site of the blue copper protein azurin. The bioconjugates showed thermosensitive behavior in electron-transfer processes with reduced cytochrome c.

Rational engineering of Thermus thermophilus cytochrome c552 to a thermally tolerant artificial peroxidase

In a previous study, we constructed a prototype thermally tolerant artificial peroxidase from an electron transfer protein, cytochrome c(552) from Thermus thermophilus, and demonstrated that engineering of proteins from thermophiles could be a promising methodology to produce artificial enzymes for practical use. In the present study, further improvement of the prototype (the V49D/M69A mutant) in enzymatic activity and thermal tolerance has been achieved by successive modifications based on detailed analyses of an active intermediate formed in the peroxidase reaction. Spectroscopic studies revealed that the major active intermediate of V49D/M69A was an oxo-ferryl heme with a protein radical predominantly localized on Tyr45. The magnetic power saturation measurement in EPR studies showed little magnetic coupling between the oxo-ferryl heme and the tyrosyl radical. This result indicated that the oxo-ferryl heme and the tyrosyl radical served as isolated oxidants. Kinetics studies indicated that the isolated oxo-ferryl heme component in the active intermediate could be a precursor to heme degradation by the reaction with H(2)O(2). Replacement of Tyr45 with phenylalanine on V49D/M69A resulted in delocalization of the radical over the protein and increased magnetic coupling between the oxo-ferryl heme and the protein radical in the intermediate. The stronger magnetic coupling between the oxo-ferryl heme and the radical was achieved by replacement of Tyr45 with tryptophan, which was similar to a tryptophanyl radical found in active intermediates of some catalase-peroxidases. The protein radical intermediates of the Tyr45 mutants exhibited relatively higher reactivity to an organic substrate than H(2)O(2) in comparison to the basal mutant, V49D/M69A, which was preferable to suppress the heme degradation process. This was reflected in the improved enzymatic activity and thermal tolerance observed for the Tyr45 mutants in the peroxidase reaction.