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Biomedical subjects

Toshiyuki Kohno

Publications and source records attributed to Toshiyuki Kohno.

At least 19 recordsLinked to original sources
PubMed2007

RNA recognition mechanism of the minimal active domain of the human immunodeficiency virus type-2 nucleocapsid protein.

NCp8 of HIV-2 contains two CCHC-type zinc fingers connected by a linker, and is involved in many critical steps of the virus life cycle. It was previously shown that the first zinc finger flanked by the linker is the minimal active domain for specific binding to viral RNA. In our previous study, we determined the three-dimensional structure of NCp8-f1, including the minimal active domain, and found that a hydrogen bond between Asn(11) N(delta)H and Arg(27) O stabilized the conformation of the linker in the vicinity of the zinc finger [Kodera et al. (1998) Biochemistry 37, 17704-17713]. In this study, RNA binding activities of NCp8-f1 and three types of its mutant peptides were analysed by native PAGE assay. The activity and three-dimensional structure of NCp8-f1/N11A, in which alanine is substituted for Asn(11) thereby affecting the conformation of the linker, was analyzed and compared with those of NCp8-f1. We demonstrated that the existence of Arg(4) and/or Lys(5) and Arg(26) and/or Arg(27) were necessary for binding RNA. Furthermore, the linker's flexible orientation, which is controlled by the hydrogen bond between Asn(11) N(delta)H and Arg(27) O, appears to be a structural basis for NCp8 existing as a multi-functional protein.

Capsid Proteins↗
PubMed2006

Trivalent recognition unit of innate immunity system: crystal structure of trimeric human M-ficolin fibrinogen-like domain.

Ficolins are a kind of pathogen-recognition molecule in the innate immune systems. To investigate the discrimination mechanism between self and non-self by ficolins, we determined the crystal structure of the human M-ficolin fibrinogen-like domain (FD1), which is the ligand-binding domain, at 1.9A resolution. Although the FD1 monomer shares a common fold with the fibrinogen gamma fragment and tachylectin-5A, the Asp-282-Cys-283 peptide bond, which is the predicted ligand-binding site on the C-terminal P domain, is a normal trans bond, unlike the cases of the other two proteins. The trimeric formation of FD1 results in the separation of the three P domains, and the spatial arrangement of the three predicted ligand-binding sites on the trimer is very similar to that of the trimeric collectin, indicating that such an arrangement is generally required for pathogen-recognition. The ligand binding study of FD1 in solution indicated that the recombinant protein binds to N-acetyl-d-glucosamine and the peptide Gly-Pro-Arg-Pro and suggested that the ligand-binding region exhibits a conformational equilibrium involving cis-trans isomerization of the Asp-282-Cys-283 peptide bond. The crystal structure and the ligand binding study of FD1 provide an insight of the self- and non-self discrimination mechanism by ficolins.

Binding Sites↗
PubMed2006

Crystallization and preliminary crystallographic analysis of a novel plant type III polyketide synthase that produces pentaketide chromone.

Pentaketide chromone synthase (PCS) from Aloe arborescens is a novel plant-specific type III polyketide synthase that catalyzes the formation of 5,7-dihydroxy-2-methylchromone from five molecules of malonyl-CoA. Recombinant PCS expressed in Escherichia coli was crystallized by the hanging-drop vapour-diffusion method. The crystals belonged to space group P2(1), with unit-cell parameters a = 73.2, b = 88.4, c = 70.0 A, alpha = gamma = 90.0, beta = 95.6 degrees . Diffraction data were collected to 1.6 A resolution using synchrotron radiation at BL24XU of SPring-8.

Aloe↗
PubMed2006

Experimental determination of particle range and dose distribution in thick targets through fragmentation reactions of stable heavy ions.

In radiation therapy with highly energetic heavy ions, the conformal irradiation of a tumour can be achieved by using their advantageous features such as the good dose localization and the high relative biological effectiveness around their mean range. For effective utilization of such properties, it is necessary to evaluate the range of incident ions and the deposited dose distribution in a patient's body. Several methods have been proposed to derive such physical quantities; one of them uses positron emitters generated through projectile fragmentation reactions of incident ions with target nuclei. We have proposed the application of the maximum likelihood estimation (MLE) method to a detected annihilation gamma-ray distribution for determination of the range of incident ions in a target and we have demonstrated the effectiveness of the method with computer simulations. In this paper, a water, a polyethylene and a polymethyl methacrylate target were each irradiated with stable (12)C, (14)N, (16)O and (20)Ne beams. Except for a few combinations of incident beams and targets, the MLE method could determine the range of incident ions R(MLE) with a difference between R(MLE) and the experimental range of less than 2.0 mm under the circumstance that the measurement of annihilation gamma rays was started just after the irradiation of 61.4 s and lasted for 500 s. In the process of evaluating the range of incident ions with the MLE method, we must calculate many physical quantities such as the fluence and the energy of both primary ions and fragments as a function of depth in a target. Consequently, by using them we can obtain the dose distribution. Thus, when the mean range of incident ions is determined with the MLE method, the annihilation gamma-ray distribution and the deposited dose distribution can be derived simultaneously. The derived dose distributions in water for the mono-energetic heavy-ion beams of four species were compared with those measured with an ionization chamber. The good agreement between the derived and the measured distributions implies that the deposited dose distribution in a target can be estimated from the detected annihilation gamma-ray distribution with a positron camera.

Algorithms↗
PubMed2006

Application of heavy-ion CT.

In treatment planning for hadron therapy, information about the relative stopping power in a patient's body is used to calculate the range of incident ions. This information is obtained from computed tomography (CT) images using a conversion table from x-ray CT numbers into stopping powers relative to the stopping power of water. In treatment planning at the National Institute of Radiological Sciences (NIRS), the conversion table has been created based on the polybinary tissue model. However, it has not been fully verified that the model is accurate enough for use in real animal tissues. In order to irradiate heavy ions more precisely in radiotherapy, we have to evaluate the accuracy of the polybinary tissue calibration in animal tissues. We have measured animal tissue samples with a heavy-ion CT (HICT) and an x-ray CT. The x-ray CT image was converted to an image of relative stopping power by using the table derived from the polybinary tissue calibration (polybinary-tissue-model CT (PTCT) image). On the other hand, with HICT, the two-dimensional distribution of relative stopping power can be obtained directly. A comparison between PTCT and HICT images enabled us to verify the accuracy of the conversion table derived from the polybinary tissue calibration. Consequently, it was found that the agreement between the relative stopping powers of PTCT and HICT is 1.6% for fat, muscle and bone.

Animals↗
PubMed2006

Overexpression, purification and preliminary crystallographic analysis of human M-ficolin fibrinogen-like domain.

Ficolins, which are comprised of a collagen-like domain and a fibrinogen-like domain, are a kind of pattern-recognition molecule for pathogens in the innate immunity system. To investigate the molecular mechanism of the discrimination between self and non-self by ficolins, human M-ficolin fibrinogen-like domain (FD1), which contains the ligand-binding site, was overexpressed in Pichia pastoris, purified and crystallized using the vapour-diffusion method at 293 K. The crystals belong to the monoclinic space group P2(1), with unit-cell parameters a = 55.16, b = 117.45, c = 55.19 angstroms, beta = 99.88 degrees, and contain three molecules per asymmetric unit. An X-ray data set was collected to 1.9 angstroms resolution using synchrotron radiation at beamline BL24XU at the SPring-8 facility in Japan.

Crystallography, X-Ray↗
PubMed2006

Structure of tightly membrane-bound mastoparan-X, a G-protein-activating peptide, determined by solid-state NMR.

The structure of mastoparan-X (MP-X), a G-protein activating peptide from wasp venom, in the state tightly bound to anionic phospholipid bilayers was determined by solid-state NMR spectroscopy. Carbon-13 and nitrogen-15 NMR signals of uniformly labeled MP-X were completely assigned by multidimensional intraresidue C-C, N-CalphaCbeta, and N-Calpha-C', and interresidue Calpha-CalphaCbeta, N-CalphaCbeta, and N-C'-Calpha correlation experiments. The backbone torsion angles were predicted from the chemical shifts of 13C', 13Calpha, 13Cbeta, and 15N signals with the aid of protein NMR database programs. In addition, two 13C-13C and three 13C-15N distances between backbone nuclei were precisely measured by rotational resonance and REDOR experiments, respectively. The backbone structure of MP-X was determined from the 26 dihedral angle restraints and five distances with an average root-mean-square deviation of 0.6 A. Peptide MP-X in the bilayer-bound state formed an amphiphilic alpha-helix for residues Trp3-Leu14 and adopted an extended conformation for Asn2. This membrane-bound conformation is discussed in relation to the peptide's activities to form pores in membranes and to activate G-proteins. This study demonstrates the power of multidimensional solid-state NMR of uniformly isotope-labeled molecules and distance measurements for determining the structures of peptides bound to lipid membranes.

GTP-Binding Proteins↗
PubMed2006

Engineered biosynthesis of plant polyketides: manipulation of chalcone synthase.

[reaction: see text]. Chalcone synthase (CHS) is a plant-specific type III polyketide synthase catalyzing condensation of 4-coumaroyl-CoA with three molecules of malonyl-CoA. Surprisingly, it was demonstrated that S338V mutant of Scutellaria baicalensis CHS produced octaketides SEK4/SEK4b from eight molecules of malonyl-CoA. Further, the octaketides-forming activity was dramatically increased in a CHS triple mutant (T197G/G256L/S338T). The functional conversion is based on the simple steric modulation of a chemically inert residue lining the active-site cavity.

Acyltransferases↗
PubMed2005

Simulation for position determination of distal and proximal edges for SOBP irradiation in hadron therapy by using the maximum likelihood estimation method.

In radiation therapy with hadron beams, conformal irradiation to a tumour can be achieved by using the properties of incident ions such as the high dose concentration around the Bragg peak. For the effective utilization of such properties, it is necessary to evaluate the volume irradiated with hadron beams and the deposited dose distribution in a patient's body. Several methods have been proposed for this purpose, one of which uses the positron emitters generated through fragmentation reactions between incident ions and target nuclei. In the previous paper, we showed that the maximum likelihood estimation (MLE) method could be applicable to the estimation of beam end-point from the measured positron emitting activity distribution for mono-energetic beam irradiations. In a practical treatment, a spread-out Bragg peak (SOBP) beam is used to achieve a uniform biological dose distribution in the whole target volume. Therefore, in the present paper, we proposed to extend the MLE method to estimations of the position of the distal and proximal edges of the SOBP from the detected annihilation gamma ray distribution. We confirmed the effectiveness of the method by means of simulations. Although polyethylene was adopted as a substitute for a soft tissue target in validating the method, the proposed method is equally applicable to general cases, provided that the reaction cross sections between the incident ions and the target nuclei are known. The relative advantage of incident beam species to determine the position of the distal and the proximal edges was compared. Furthermore, we ascertained the validity of applying the MLE method to determinations of the position of the distal and the proximal edges of an SOBP by simulations and we gave a physical explanation of the distal and the proximal information.

Algorithms↗
PubMed2005

Direct determination of a membrane-peptide interface using the nuclear magnetic resonance cross-saturation method.

Membrane-peptide interactions are involved in many crucial biological and pharmacological activities. To clarify the interaction mode of membrane-peptide complexes, it is important to analyze both the dynamic properties and the contact residues of the membrane-bound peptide. In this study, we investigated the dynamic properties of a peptide bound to a lipid bilayer, using relaxation and amide-water exchange analyses, and directly determined the membrane-peptide interface, using the cross-saturation method. For the models of a lipid bilayer and a peptide, isotropic bicelles and mastoparan were used, respectively. The results indicate that mastoparan had a heterogeneous distribution of motion over various timescales and interacted with the lipid bilayer by using its hydrophobic side; the molecule was located within the lipid bilayer rather than on the surface, as thought previously. This study shows that the cross-saturation method is useful for determining the interface of not only protein-protein but also membrane-peptide complexes.

Algorithms↗
PubMed2005

Spatial fragment distribution from a therapeutic pencil-like carbon beam in water.

The latest heavy ion therapy tends to require information about the spatial distribution of the quality of radiation in a patient's body in order to make the best use of any potential advantage of swift heavy ions for the therapeutic treatment of a tumour. The deflection of incident particles is described well by Molière's multiple-scattering theory of primary particles; however, the deflection of projectile fragments is not yet thoroughly understood. This paper reports on our investigation of the spatial distribution of fragments produced from a therapeutic carbon beam through nuclear reactions in thick water. A DeltaE-E counter telescope system, composed of a plastic scintillator, a gas-flow proportional counter and a BGO scintillator, was rotated around a water target in order to measure the spatial distribution of the radiation quality. The results revealed that the observed deflection of fragment particles exceeded the multiple scattering effect estimated by Molière's theory. However, the difference can be sufficiently accounted for by considering one term involved in the multiple-scattering formula; this term corresponds to a lateral 'kick' at the point of production of the fragment. This kick is successfully explained as a transfer of the intra-nucleus Fermi momentum of a projectile to the fragment; the extent of the kick obeys the expectation derived from the Goldhaber model.

Carbon↗
PubMed2005

Responses of a diamond detector to high-LET charged particles.

The responses of a commercial diamond detector (type 60003, PTW-Freiburg) to several heavy ions were examined. The responses to heavy-ion beams reached stable levels with relatively small pre-irradiation doses compared to photon-beam irradiations. The responses also reached stable levels with a smaller pre-irradiation dose when the dose rate of the He beams was increased. A total accumulated dose of about 5 Gy was required for the pre-irradiation dose of heavy-ion beams. No angular dependence of the detector responses was observed within a deviation of 5%. The dose-rate dependence of the detector responses to heavy-ion beams was far smaller than that to gamma rays. The decrease in the response was within 0.9%, with a variation from 0.88 to 18.2 Gy min(-1) in the carbon beam. We examined the LET dependence of the diamond detector responses using various kinds of heavy-ion beams. The responses had particle dependence in addition to LET dependence. The responses decreased more with higher LET particles and decreased less with large-Z particles. We proposed a gradual-saturation model based on the track structure under several simple assumptions to explain the LET and particle dependences of the response.

Diamond↗
PubMed2005

Quantitative comparison of suitability of various beams for range monitoring with induced beta+ activity in hadron therapy.

In radiation therapy with hadron beams, it is important to evaluate the range of incident ions and the deposited dose distribution in a patient body for the effective utilization of such properties as the dose concentration and the biological effect around the Bragg peak. However, there is some ambiguity in determining this range because of a conversion error from the x-ray CT number to the charged particle range. This is because the CT number is related to x-ray absorption coefficients, while the ion range is determined by the electron density of the substance. Using positron emitters produced in the patient body through fragmentation reactions during the irradiation has been proposed to overcome this problem. The activity distribution in the patient body can be deduced by detecting pairs of annihilation gamma rays emitted from the positron emitters, and information about the range of incident ions can be obtained. In this paper, we propose a quantitative comparison method to evaluate the mean range of incident ions and monitor the activity distribution related to the deposited dose distribution. The effectiveness of the method was demonstrated by evaluating the range of incident ions using the maximum likelihood estimation (MLE) method and Fisher's information was calculated under realistic conditions for irradiations with several kinds of ions. From the calculated Fisher's information, we compared the relative advantages of initial beams to determine the range of incident ions. The (16)O irradiation gave the most information among the stable heavy ions when we measured the induced activity for 500 s and 60 s just after the irradiation. Therefore, under these conditions, we concluded that the (16)O beam was the optimum beam to monitor the activity distribution and to evaluate the range. On the other hand, if the positron emitters were injected directly as a therapeutic beam, the (15)O irradiation gave the most information. Although the relative advantages of initial beams as well as the measured activity distributions slightly varied according to the measurement conditions, comparisons could be made for different conditions by using Fisher's information.

Algorithms↗
PubMed2005

Production of proteins for NMR studies using the wheat germ cell-free system.

This chapter describes protocols for preparing 15N-labeled proteins (ubiquitin is used as an example) using Escherichia coli cells (with purification) and the wheat germ cell-free system (without purification). A comparison of 1H-15N heteronuclear single-quantum coherence (HSQC) spectra of yeast ubiquitin prepared using each method indicates that this wheat germ cell-free system may be used for rapid nuclear magnetic resonance analyses of proteins without purification.

Cell-Free System↗
PubMed2004

Paramagnetic NMR study of Cu(2+)-IDA complex localization on a protein surface and its application to elucidate long distance information.

The paramagnetic metal chelate complex Cu(2+)-iminodiacetic acid (Cu(2+)-IDA) was mixed with ubiquitin, a small globular protein. Quantitative analyses of (1)H and (15)N chemical shift changes and line broadenings induced by the paramagnetic effects indicated that Cu(2+)-IDA was localized to a histidine residue (His68) on the ubiquitin surface. The distances between the backbone amide proton and the Cu(2+) relaxation center were evaluated from the proton transverse relaxation rates enhanced by the paramagnetic effect. These correlated well with the distances calculated from the crystal structure up to 20 A. Here, we show that a Cu(2+)-IDA is the first paramagnetic reagent that specifically localizes to a histidine residue on the protein surface and gives the long-range distance information.

Amides↗
PubMed2004

Structure of human translin at 2.2 A resolution.

The structure of human translin at 2.2 A resolution is reported in space group C222(1). Translin forms a tetramer in the asymmetric unit. Although the monomer structure is almost the same as the crystal structure of murine translin in space group P2(1)2(1)2, the relative positions of the tetramers differ between the human and murine translins. This suggests that the multimerization of translin is flexible; the flexibility may be related to the binding to DNA/RNA.

Crystallization↗