Biomedical subjects

Takeshi Todo

Publications and source records attributed to Takeshi Todo.

At least 19 recordsLinked to original sources

PubMed2006

Autonomic and cardiovascular responses to scent stimulation are altered in cry KO mice.

Previously, we observed that in rats, olfactory stimulation with scent of grapefruit oil (SGFO) elevates the activities of sympathetic nerves. SGFO also suppresses gastric vagal (parasympathetic) nerve activity (GVNA), increases the plasma glycerol concentration, blood pressure (BP) and body temperature, and reduces appetite. In contrast, olfactory stimulation with scent of lavender oil (SLVO) has opposite effects in rats. Here, we show that in mice, olfactory stimulation with SGFO elevated activities of sympathetic nerves innervating the kidney, adrenal gland and brown adipose tissue as well as increasing BP and suppressing GVNA, whereas olfactory stimulation with SLVO decreased these sympathetic nerve activities and BP, and elevated GVNA. Electrolytic lesions of the mouse hypothalamic suprachiasmatic nucleus (SCN) eliminated changes in renal sympathetic nerve activity (RSNA), BP and GVNA induced by either SGFO or SLVO. Furthermore, SGFO-induced elevations in RSNA and BP and the SLVO-induced GVNA increase were not observed in Cryptochrome (Cry)-deficient mice, which harbor mutations in both cry1 and cry2 and lack normal circadian rhythms. These findings suggest that SGFO and SLVO affect autonomic neurotransmission and BP via the SCN in mice. Moreover, the molecular clock mechanism in the SCN, which involves the cry1 and cry2 genes, is partially involved in mediating these autonomic and cardiovascular actions of SGFO and SLVO.

Adipose Tissue, Brown↗

PubMed2006

Electron nuclear double resonance differentiates complementary roles for active site histidines in (6-4) photolyase.

(6-4) photolyase catalyzes the light-dependent repair of UV-damaged DNA containing (6-4) photoproducts. Blue light excitation of the enzyme generates the neutral FAD radical, FADH., which is believed to be transiently formed during the enzymatic DNA repair. Here (6-4) photolyase has been examined by optical spectroscopy, electron paramagnetic resonance, and pulsed electron nuclear double resonance spectroscopy. Characterization of selected proton hyperfine couplings of FADH., namely those of H(8alpha) and H(1'), yields information on the micropolarity at the site where the DNA substrate is expected to bind. Shifts in the hyperfine couplings as a function of structural modifications induced by point mutations and pH changes distinguish the protonation states of two highly conserved histidines, His(354) and His(358), in Xenopus laevis (6-4) photolyase. These are proposed to catalyze formation of the oxetane intermediate that precedes light-initiated DNA repair. The results show that at pH 9.5, where the enzymatic repair activity is highest, His(358) is deprotonated, whereas His(354) is protonated. Hence, the latter is likely the proton donor that initiates oxetane formation from the (6-4) photoproduct.

Animals↗

PubMed2006

Molecular analysis of Dec1 and Dec2 in the peripheral circadian clock of zebrafish photosensitive cells.

To elucidate the roles of DEC1 and DEC2, basic helix-loop-helix transcription factors, in the circadian clock of photosensitive zebrafish peripheral cells, zebrafish Dec1 and Dec2 (zDec1 and zDec2) were cloned and their functions and expression patterns were examined in BRF41, a zebrafish cell line. zDEC1 and zDEC2 have high sequence similarity to mammalian counterparts and the molecular phylogenetic analysis of the zDEC1 and zDEC2 sequences reflected the predicted pattern of species classification. zDEC1 and zDEC2 inhibited zCLOCK1:zBMAL3 mediated transcription as CRY1a. zDec1 and zDec2 mRNA showed robust circadian oscillation in BRF41 cells. However, zDec1 and zDec2 mRNA was not strongly induced by exposure to light. These results indicate that zDec1 and zDec2 are involved in the circadian clock mechanism in photosensitive zebrafish peripheral cells by suppressing CLOCK/BMAL-induced gene expression and that the feedback loops of zDEC1 and zDEC2 may be interlocked with the PER/CRY core circadian feedback loops.

Animals↗

PubMed2006

Photoreaction cycle of the light, oxygen, and voltage domain in FKF1 determined by low-temperature absorption spectroscopy.

Flavin-binding Kelch repeat F-box (FKF1) protein plays important roles in the photoregulation of flowering in Arabidopsis. FKF1 has a light, oxygen, and voltage (LOV) sensing domain binding a flavin mononucleotide (FMN) as a chromophore noncovalently. Photoreaction of the FKF1-LOV polypeptide was studied by low-temperature absorption spectroscopy. Upon blue light irradiation, a ground state, D(450), is converted to S(390) known as a cysteinyl-flavin adduct intermediate in the photoreaction of phototropin. Below 150 K, bleaching of D(450) was much reduced and a new photoproduct, Z(370), appeared as well as S(390) formation. The calculated absorption spectrum for Z(370) is very similar to those of flavoproteins in an anion radical state. On the basis of the results that S(390) formation proceeds to Z(370) formation and that Z(370) formed at low temperatures reverts to D(450) upon temperature increase, Z(370) is concluded to be not an intermediate from D(450) to S(390). Z(370) is suggested to be formed from the biradical triplet-excited state after relaxing to the ground state with the FMN anion radical trapped at the low temperature, in which the SH of the cysteine is in the wrong position that is able to produce a radical pair but unable to form the cysteinyl-flavin adduct. The counter SH in the cationic radical state may revert to the ground state by extracting an electron from the unidentified amino acid residue. Interestingly, S(390) that has been thought to be irreversible to D(450) was revealed to revert to D(450) very slowly with a half-life time of 62.5 h in solution at 298 K. The photoreaction mechanism is discussed in reference to the calculated activation energy of the reaction processes.

Amino Acid Sequence↗

PubMed2006

Chemical synthesis of oligodeoxyribonucleotides containing the Dewar valence isomer of the (6-4) photoproduct and their use in (6-4) photolyase studies.

The pyrimidine(6-4)pyrimidone photoproduct, a major UV lesion formed between adjacent pyrimidine bases, is transformed to its Dewar valence isomer upon exposure to UVA/UVB light. We have synthesized a phosphoramidite building block of the Dewar photoproduct formed at the thymidylyl(3'-5')thymidine site and incorporated it into oligodeoxyribonucleotides. The diastereoisomers of the partially protected dinucleoside monophosphate bearing the (6-4) photoproduct, which were caused by the chirality of the phosphorus atom, were separated by reversed-phase chromatography, and the (6-4) photoproduct was converted to the Dewar photoproduct by irradiation of each isomer with Pyrex-filtered light from a high-pressure mercury lamp. The Dewar photoproduct was stable under both acidic and alkaline conditions at room temperature. After characterization of the isomerized base moiety by NMR spectroscopy, a phosphoramidite building block was synthesized in three steps. Although the ordinary method could be used for the oligonucleotide synthesis, benzimidazolium triflate as an alternative activator yielded better results. The oligonucleotides were used for the analysis of the reaction and the binding of Xenopus (6-4) photolyase. Although the affinity of this enzyme for the Dewar photoproduct-containing duplex was reportedly similar to that for the (6-4) photoproduct-containing substrate, the results suggested a difference in the binding mode.

Animals↗

PubMed2006

Characteristic structure and environment in FAD cofactor of (6-4) photolyase along function revealed by resonance Raman spectroscopy.

A pyrimidine-pyrimidone (6-4) photoproduct and a cyclobutane pyrimidine dimer (CPD) are major DNA lesions induced by ultraviolet irradiation, and (6-4) photolyase, an enzyme with flavin adenine dinucleotide (FAD) as a cofactor, repairs the former specifically by light illumination. We investigated resonance Raman spectra of (6-4) photolyase from Arabidopsis thaliana having neutral semiquinoid and oxidized forms of FAD, which were selectively intensity enhanced by excitations at 568.2 and 488.0 nm, respectively. DFT calculations were carried out for the first time on the neutral semiquinone. The marker band of a neutral semiquinone at 1606 cm(-1) in H(2)O, whose frequency is the lowest among various flavoenzymes, apparently splits into two comparable bands at 1594 and 1608 cm(-1) in D(2)O, and similarly, that at 1522 cm(-1) in H(2)O does into three bands at 1456, 1508, and 1536 cm(-1) in D(2)O. This D(2)O effect was recognized only after being oxidized once and photoreduced to form a semiquinone again, but not by simple H/D exchange of solvent. Some Raman bands of the oxidized form were observed at significantly low frequencies (1621, 1576 cm(-1)) and with band splittings (1508/1493, 1346/1320 cm(-1)). These Raman spectral characteristics indicate strong H-bonding interactions (at N5-H, N1), a fairly hydrophobic environment, and an electron-lacking feature in benzene ring of the FAD cofactor, which seems to specifically control the reactivity of (6-4) photolyase.

Amino Acid Sequence↗

PubMed2006

Similarities and differences between cyclobutane pyrimidine dimer photolyase and (6-4) photolyase as revealed by resonance Raman spectroscopy: Electron transfer from the FAD cofactor to ultraviolet-damaged DNA.

The cyclobutane pyrimidine dimer (CPD) and (6-4) photoproduct, two major types of DNA damage caused by UV light, are repaired under illumination with near UV-visible light by CPD and (6-4) photolyases, respectively. To understand the mechanism of DNA repair, we examined the resonance Raman spectra of complexes between damaged DNA and the neutral semiquinoid and oxidized forms of (6-4) and CPD photolyases. The marker band for a neutral semiquinoid flavin and band I of the oxidized flavin, which are derived from the vibrations of the benzene ring of FAD, were shifted to lower frequencies upon binding of damaged DNA by CPD photolyase but not by (6-4) photolyase, indicating that CPD interacts with the benzene ring of FAD directly but that the (6-4) photoproduct does not. Bands II and VII of the oxidized flavin and the 1398/1391 cm(-1) bands of the neutral semiquinoid flavin, which may reflect the bending of U-shaped FAD, were altered upon substrate binding, suggesting that CPD and the (6-4) photoproduct interact with the adenine ring of FAD. When substrate was bound, there was an upshifted 1528 cm(-1) band of the neutral semiquinoid flavin in CPD photolyase, indicating a weakened hydrogen bond at N5-H of FAD, and band X seemed to be downshifted in (6-4) photolyase, indicating a weakened hydrogen bond at N3-H of FAD. These Raman spectra led us to conclude that the two photolyases have different electron transfer mechanisms as well as different hydrogen bonding environments, which account for the higher redox potential of CPD photolyase.

Arabidopsis↗

PubMed2006

Synthesis of oligonucleotides containing the Dewar valence isomer of the (6-4) photoproduct and their application to (6-4) photolyase studies.

The (6-4) photoproduct, which is a major UV light-induced lesion formed between adjacent pyrimidine bases, is isomerized to its Dewar valence isomer by exposure to longer wavelengths. We have synthesized a phosphoramidite building block of the Dewar photoproduct formed at the thymidylyl(3'-5')thymidine site, and incorporated it into oligonucleotides on a DNA synthesizer, aiming to use them for biological studies. An alternative activator, benzimidazolium triflate, gave better results, while by-products were detected at longer retention time in the ordinary synthesis. We characterized the synthetic oligonucleotides by UV conversion, nuclease digestion and mass spectrometry. Their use in the study of the (6-4) photolyase, a DNA repair enzyme, revealed its different recognition modes between the (6-4) photoproduct and the Dewar isomer.

Chromatography, High Pressure Liquid↗

PubMed2006

Circadian intraocular pressure rhythm is generated by clock genes.

PURPOSE: The present study in a mouse model was undertaken to reveal the role of the circadian clock genes Cry1 and Cry2 in generation of 24-hour intraocular pressure (IOP) rhythm. METHODS: IOP was measured at eight time points daily (circadian time [CT] 0, 3, 6, 9, 12, 15, 18, and 21 hours), using a microneedle method in four groups of C57BL/6J mice (groups 1 and 3, wild-type; groups 2 and 4, Cry-deficient [Cry1-/-Cry2-/-]). During the IOP measurements, mice in groups 1 and 2 were maintained in a 12-hour light-dark cycle (LD), whereas mice in groups 3 and 4 were kept in a constant darkness (DD) that started 24 to 48 hours before the measurements. Circadian IOP variations in each group were evaluated by one-way analysis of variance (ANOVA) and Scheffé tests. RESULTS: In wild-type mice living in LD conditions, pressures measured in the light phase were significantly lower than those in the dark phase. This daily rhythm was maintained under DD conditions with low pressure in the subjective day and high pressure in the subjective night. In contrast, Cry-deficient mice did not show significant circadian changes in IOP, regardless of environmental light conditions. CONCLUSIONS: These findings demonstrate that clock oscillatory mechanisms requiring the activity of core clock genes are essential for the generation of a circadian rhythm of intraocular pressure.

Animals↗

PubMed2006

Generation of medaka gene knockout models by target-selected mutagenesis.

We have established a reverse genetics approach for the routine generation of medaka (Oryzias latipes) gene knockouts. A cryopreserved library of N-ethyl-N-nitrosourea (ENU) mutagenized fish was screened by high-throughput resequencing for induced point mutations. Nonsense and splice site mutations were retrieved for the Blm, Sirt1, Parkin and p53 genes and functional characterization of p53 mutants indicated a complete knockout of p53 function. The current cryopreserved resource is expected to contain knockouts for most medaka genes.

Adenosine Triphosphatases↗

PubMed2005

The cryptochromes.

Cryptochromes are photoreceptors that regulate entrainment by light of the circadian clock in plants and animals. They also act as integral parts of the central circadian oscillator in animal brains and as receptors controlling photomorphogenesis in response to blue or ultraviolet (UV-A) light in plants. Cryptochromes are probably the evolutionary descendents of DNA photolyases, which are light-activated DNA-repair enzymes, and are classified into three groups -- plant cryptochromes, animal cryptochromes, and CRY-DASH proteins. Cryptochromes and photolyases have similar three-dimensional structures, characterized by an alpha/beta domain and a helical domain. The structure also includes a chromophore, flavin adenine dinucleotide (FAD). The FAD-access cavity of the helical domain is the catalytic site of photolyases, and it is predicted also to be important in the mechanism of cryptochromes.

Animals↗

PubMed2005

Reduced alpha-adrenoceptor responsiveness and enhanced baroreflex sensitivity in Cry-deficient mice lacking a biological clock.

To reveal the role of clock genes in generating the circadian rhythm of baroreflexes, we continuously measured mean arterial pressure and baroreflex sensitivity in free-moving normal wild-type mice, and in Cry-deficient mice which lack a circadian rhythm, in constant darkness for 24 h. In wild-type mice the mean arterial pressure was higher at night than during the day, and was accompanied by a significantly enhanced baroreflex sensitivity of -13.6 +/- 0.8 at night compared with -9.7 +/- 0.7 beats min(-1) mmHg(-1) during the day (P < 0.001). On the other hand, diurnal changes in arterial pressure disappeared in Cry-deficient mice with remarkably enhanced baroreflex sensitivity compared with wild-type mice (P < 0.001): -21.9 +/- 1.6 at night and -23.1 +/- 2.1 beats min(-1) mmHg(-1) during the day. Moreover, the mean arterial pressure response to 10 microg kg(-1) of phenylephrine, an alpha1-adrenoceptor agonist, was severely suppressed in Cry-deficient mice regardless of time, while that for the wild-type mice was 10.1 +/- 1.9 mmHg in the night, significantly lower than 22.0 +/- 3.5 mmHg in the day (P < 0.01). These results suggest that CRY genes are involved in generating the circadian rhythm of baroreflex sensitivity, partially by regulating alpha(1)-adrenoceptor-mediated vasoconstriction in peripheral vessels.

Adrenergic alpha-Agonists↗

PubMed2005

Determination of the g-matrix orientation in flavin radicals by high-field/high-frequency electron-nuclear double resonance.

A high-microwave-frequency/high-magnetic-field pulsed electron-nuclear double resonance (ENDOR) study performed at 94 GHz on the flavin semiquinone cofactor of Xenopus laevis (6-4) photolyase in its neutral radical state is presented. Although the principal values of the flavin radical's g-matrix are not fully resolved in the 94-GHz EPR spectrum in a nonoriented sample, the orientation of the principal axes of g is obtained by exploiting the orientation selection of the proton ENDOR signals from the methyl protons at C-8alpha and the deuteron ENDOR signals from D-5 in an enzyme sample in deuterated buffer. This procedure for assigning the orientation of g relative to the molecular frame makes use of commercially available ENDOR instrumentation without the necessity to perform single-crystal studies.

Animals↗

PubMed2004

Role of Gln1029 in the photoactivation processes of the LOV2 domain in adiantum phytochrome3.

Phototropin (phot) is a blue-light receptor in plants. The molecule has two FMN (flavin mononucleotide)-binding domains named the LOV (light-oxygen-voltage) domain, that is a subset of a PAS (per-arnt-sim) superfamily. Illumination of phot-LOV domains produces a covalent C(4a) flavin-cysteinyl adduct, which is called the S390 intermediate state. According to the crystal structures of the LOV2 domain of Adiantum phytochrome3 (phy3), a fusion protein of phot containing the phytochrome chromophoric domain, in the unphotolyzed and S390 states, and the side chain of Gln1029 switches hydrogen bonds with the FMN chromophore. Gln1029 is the hydrogen-bonding donor of the C(4)=O group of FMN in the unphotolyzed state, whereas Gln1029 is the hydrogen-bonding acceptor of the N(5)-H group of FMN in S390. In this paper, we measured the light-induced structural changes in the Q1029L mutant protein of phy3-LOV2 by means of low-temperature FTIR spectroscopy, and the obtained spectra are compared with those of the wild type. Low-temperature UV-visible spectroscopy of Q1029L detected only one intermediate state, S390, at 77-295 K, as well as the wild type. The C(4)=O stretch of FMN at 1710 cm(-1) is shifted to 1723 cm(-1) in Q1029L, presumably because of the lack of hydrogen bonds between Gln1029 and FMN. Upon formation of S390, the C(4)=O group hydrogen bond is weakened in both wild type and Q1029L. These observations are fully consistent with the X-ray crystal structures of the unphotolyzed and S390 states. On the other hand, the C(4)=O stretch of FMN and amide-I vibrations are temperature-independent in Q1029L, in contrast to wild type, in which highly temperature-dependent FTIR spectra are detected. Amide-I vibrations of Q1029L at room temperature are similar to those of the wild type at 77-150 K but not at room temperature. These facts imply that the Q1029L mutant protein lacks progressive protein structural changes following flavin-cysteinyl adduct formation in the wild type, which eventually alter structures of beta sheet and alpha helix in the protein moiety. Hydrogen-bonding interaction of Gln1029 with the FMN chromophore likely plays an important role in the protein structural changes of phy3-LOV2.

Adiantum↗

PubMed2004

Assay method for Escherichia coli photolyase activity using single-strand cis-syn cyclobutane pyrimidine dimer DNA as substrate.

A high-performance liquid chromatography method for the assay of Escherichia coli photolyase activity was developed. When cis-syn cyclobutane pyrimidine dimer was used as substrate, the Michaelis constant (K(m)) value for the photolyase activity was 100 nM. The linear range of the calibration curve of the photolyase activity was 0.026-6.64 microU/assay tube. The correlation coefficient for this linearity was 0.998. The limit of detection (S/N = 3) was 26 nU/assay tube. The photolyase activity was increased 1.6-fold in the presence of 5,10-methenyltetrahydrofolic acid in the enzyme reaction mixture.

Chromatography, High Pressure Liquid↗

PubMed2004

Investigation of the cyclobutane pyrimidine dimer (CPD) photolyase DNA recognition mechanism by NMR analyses.

The cyclobutane pyrimidine dimer (CPD) is one of the major forms of DNA damage caused by irradiation with ultraviolet (UV) light. CPD photolyases recognize and repair UV-damaged DNA. The DNA recognition mechanism of the CPD photolyase has remained obscure because of a lack of structural information about DNA-CPD photolyase complexes. In order to elucidate the CPD photolyase DNA binding mode, we performed NMR analyses of the DNA-CPD photolyase complex. Based upon results from (31)P NMR measurements, in combination with site-directed mutagenesis, we have demonstrated the orientation of CPD-containing single-stranded DNA (ssDNA) on the CPD photolyase. In addition, chemical shift perturbation analyses, using stable isotope-labeled DNA, revealed that the CPD is buried in a cavity within CPD photolyase. Finally, NMR analyses of a double-stranded DNA (dsDNA)-CPD photolyase complex indicated that the CPD is flipped out of the dsDNA by the enzyme, to gain access to the active site.

Binding Sites↗

PubMed2004

Identification of radiation-sensitive mutants in the Medaka, Oryzias latipes.

We screened populations of N-ethyl-N-nitrosourea (ENU)-mutagenized Medaka, (Oryzias latipes) for radiation-sensitive mutants to investigate the mechanism of genome stability induced by ionizing radiation in developing embryos. F3 embryos derived from male founders that were homozygous for induced the mutations were irradiated with gamma-rays at the organogenesis stage (48hpf) at a dose that did not cause malformation in wild-type embryos. We screened 2130 F2 pairs and identified three types of mutants with high incidence of radiation-induced curly tailed (ric) malformations using a low dose of irradiation. The homozygous strain from one of these mutants, ric1, which is highly fertile and easy to breed, was established and characterized related to gamma-irradiation response. The ric1 strain also showed higher incidence of malformation and lower hatchability compared to the wild-type CAB strain after gamma-irradiation at the morula and pre-early gastrula stages. We found that the decrease in hatching success after gamma-irradiation, depends on the maternal genotype at the ric1 locus. Terminal deoxynucleotidyl transferase-mediated deoxy-UTP nick end-labeling assays showed a high frequency of apoptosis in the ric1 embryos immediately after gamma-irradiation at the pre-early gastrula stage but apoptotic cells were not observed before midblastula transition (MBT). The neutral comet assay revealed that the ric1 mutant has a defect in the rapid repair of DNA double-strand breaks induced by gamma-rays. These results suggest that RIC1 is involved in the DNA double strand break repair in embryos from morula to organogenesis stages, and unrepaired DNA double strand breaks in ric1 trigger apoptosis after MBT. These results support the use of the ric1 strain for investigating various biological consequences of DNA double strand breaks in vivo and for sensitive monitoring of genotoxicity related to low dose radiation.

Animals↗

PubMed2004

A systematic genome-wide screen for mutations affecting organogenesis in Medaka, Oryzias latipes.

A large-scale mutagenesis screen was performed in Medaka to identify genes acting in diverse developmental processes. Mutations were identified in homozygous F3 progeny derived from ENU-treated founder males. In addition to the morphological inspection of live embryos, other approaches were used to detect abnormalities in organogenesis and in specific cellular processes, including germ cell migration, nerve tract formation, sensory organ differentiation and DNA repair. Among 2031 embryonic lethal mutations identified, 312 causing defects in organogenesis were selected for further analyses. From these, 126 mutations were characterized genetically and assigned to 105 genes. The similarity of the development of Medaka and zebrafish facilitated the comparison of mutant phenotypes, which indicated that many mutations in Medaka cause unique phenotypes so far unrecorded in zebrafish. Even when mutations of the two fish species cause a similar phenotype such as one-eyed-pinhead or parachute, more genes were found in Medaka than in zebrafish that produced the same phenotype when mutated. These observations suggest that many Medaka mutants represent new genes and, therefore, are important complements to the collection of zebrafish mutants that have proven so valuable for exploring genomic function in development.

Animals↗