Biomedical subjects
Publications and source records attributed to Ryo Takeuchi.
An array of novel analogues of the marine oxylipins, the manzamenones and plakoridines, have been prepared in divergent fashion using an approach modelled on a biogenetic theory. Many of the target compounds show potent inhibition of DNA polymerases alpha and beta and human terminal deoxynucleotidyl transferase (TdT).
Abasic (AP) sites are a threat to cellular viability and genomic integrity, since they impede transcription and DNA replication. In mammalian cells, DNA polymerase (pol) beta plays an important role in the repair of AP sites. However, it is known that many organisms, including Drosophila melanogaster, do not have a pol beta homologue, and it is unclear how they repair AP sites. Here, we screened for DNA polymerases that interact with the Drosophila AP endonuclease 1 homologue, Rrp1 (recombination repair protein 1), and found that Drosophila pol zeta (Dmpol zeta), DmREV3 and DmREV7 bound to Rrp1 in a protein affinity column. Rrp1 directly interacted with DmREV7 in vitro and in vivo but not with DmREV3. These findings suggest that the DNA polymerase partner for Rrp1 is Dmpol zeta and that this interaction occurs through DmREV7. Interestingly, DmREV7 bound to the N-terminal region of Rrp1, which has no known protein homologue, suggesting that this binding is a species-specific event. Moreover, DmREV7 could stimulate the AP endonuclease activity of Rrp1, but not the 3'-exonuclease activity, and form a homomultimer. DmREV3 could not incorporate nucleotides at the 5'-incised tetrahydrofran sites but did show strand displacement activity for one-nucleotide-gapped DNA, which was not influenced by either DmREV7 or Rrp1. Methyl methanesulfonate and hydrogen peroxide treatments increased mRNA levels of DmREV3 and DmREV7. On the basis of the direct interaction between DmREV7 and Rrp1, we suggest that Dmpol zeta may be involved in the repair pathway of AP sites in DNA.
[reaction: see text] [Ir(cod)Cl]2/DPPE was found to be a new catalyst for the cycloaddition of alpha,omega-diynes with monoynes to give polysubstituted benzene derivatives in high yields. Internal monoynes as well as terminal monoynes could be used. The reaction tolerates a broad range of functional groups such as alcohol, amine, alkene, ether, halogen, and nitrile. The reaction of 1,6-octadiyne derivatives with 1-alkynes gives ortho products and meta products. The regioselectivity could be controlled by the choice of ligand. The reaction with DPPE was meta selective, with meta selectivity of up to 82%. The reaction with DPPF was ortho selective, with ortho selectivity of up to 88%. We propose a mechanism to account for this regioselective cycloaddition. [Ir(cod)Cl](2)/DPPE also catalyzed the cycloaddition of alpha,omega-diynes with 2,5-dihydrofuran to give bicyclic cyclohexadiene derivatives. The reaction with 2,3-dihydrofuran and n-butyl vinyl ether gave benzene derivatives instead of cyclohexadiene derivatives. We also propose a mechanism to account for this novel aromatization that includes cleavage of the C-O bond.
The positioning or selection of a region of interest (ROI) is an essential step for the quantification of brain images. However, so long as the ROIs were manually selected, results obtained fluctuated considerably with subtle changes in their positioning. To perform an ROI analysis of the brain with improved objectivity and excellent reproducibility, we recently established fully automated ROI-based analysis software for the brain, the so-called 3DSRT and the FineSRT. 3DSRT performed by consecutive analyzing process as follows: (1) anatomical standardization using the SPM99 algorithm; (2) analysis using constant 318 ROIs divided into 12 groups (segments) on each hemisphere; (3) calculation of the area-weighted average for each of the respective 24 segments; (4) display of the results followed by the saving of respective values of the 636 ROIs (both hemispheres). The processes of the FineSRT were fundamentally similar to that of 3DSRT, but on FineSRT more precise constant 1394 ROIs (both hemispheres) corresponding to the respective cerebral convolutions were used. Our programs have been useful for objective estimation of cerebrovascular reserve, especially for follow-up studies of an infant with moyamoya disease, because we could compare the results without consideration of the patient's growth during the follow-up period.
The damaged DNA-binding protein (DDB) complex consists of a heterodimer of p127 (DDB1) and p48 (DDB2) subunits and is believed to have a role in nucleotide excision repair (NER). We used the GAL4-UAS targeted expression system to knock down DDB1 in wing imaginal discs of Drosophila. The knock-down was achieved in transgenic flies using over-expression of inverted repeat RNA of the D-DDB1 gene [UAS-D-DDB1(650)-dsRNA]. As a consequence of RNA interference (RNAi), the fly had a shrunken wing phenotype. The wing spot test showed induced genome instability in transgenic flies with RNAi knock-down of D-DDB1 in wing imaginal discs. When Drosophila larvae with RNAi knock-down of D-DDB1 in wing imaginal discs were treated with the chemical mutagen methyl methanesulfonate (MMS), the frequency of flies with a severely shrunken wing phenotype increased compared to non-treated transgenic flies. These results suggested that DDB1 plays a role in the response to DNA damaged with MMS and in genome stability in Drosophila somatic cells.
The eukaryotic DNA polymerase processivity factor, proliferating cell nuclear antigen, is an essential component in the DNA replication and repair machinery. In Drosophila melanogaster, we cloned a second PCNA cDNA that differs from that encoded by the gene mus209 (for convenience called DmPCNA1 in this article). The second PCNA cDNA (DmPCNA2) encoded a 255 amino acid protein with 51.7% identity to DmPCNA1, and was ubiquitously expressed during Drosophila development. DmPCNA2 was localized in nuclei as a homotrimeric complex and associated with Drosophila DNA polymerase delta and epsilonin vivo. Treatment of cells with methyl methanesulfonate or hydrogen peroxide increased the amount of both DmPCNA2 and DmPCNA1 associating with chromatin, whereas exposure to UV light increased the level of association of only DmPCNA1. Our observations suggest that DmPCNA2 may function as an independent sliding clamp of DmPCNA1 when DNA repair occurs.
Sulfoquinovosyldiacyglycerol (SQDG) has a wide range of biological activities that make it an attractive compound for the development of new drugs. Chemically synthesized beta-SQDG-C(18:0) (1,2-di-O-stearoyl-3-O-(6-deoxy-6-sulfo-beta-d-glucopyranosyl)-sn-glycerol), for example, has a potent inhibitory effect on DNA polymerases. We investigated the properties of the vesicle form of beta-SQDG-C(18:0) as the monomer has low solubility in water. The structure of the beta-SQDG-C(18:0) vesicles are highly influenced by NaCl concentration in preparation process. At low NaCl concentrations, the beta-SQDG-C(18:0) vesicles have high surface curvature and form small unilamellar vesicles. Increases in NaCl concentration, resulted in decreased surface curvature and a tendency for beta-SQDG-C(18:0) to form large multilamellar vesicles. The small unilamellar vesicles showed a potent inhibitory effect on DNA polymerase beta, whereas the large multilamellar vesicles had no such effect. We investigated further the relationship between vesicle size and activity by preparing smaller vesicles (262, 99 and 43 nm in diameter) using an extrusion technique. These smaller vesicles had a greater inhibitory effect on DNA polymerase beta activity than non-extruded vesicles. beta-SQDG-C(18:0) vesicles, especially those of small size, were effective in DNA polymerase inhibition and are expected to have high applicability in DNA polymerase study.
[reaction: see text] 1,6-Enynes reacted with monoynes to give cyclohexadiene derivatives in the presence of a catalytic amount of [Ir(cod)Cl](2)/ligand. DPPE was most suitable for cycloaddition. Diastereoselective cycloaddition was also possible. In the absence of monoynes, 1,6-enynes cycloisomerized to (Z)-1-alkylidene-2-methylenecyclopentane derivatives. DPPF was most suitable for cycloisomerization. These results are the first examples of highly Z-selective cycloisomerization.
A novel endo-exonuclease, DmGEN (Drosophila Melanogaster XPG-like endonuclease), was identified in D.melanogaster. DmGEN is composed of five exons and four introns, and the open reading frame encodes a predicted product of 726 amino acid residues with a molecular weight of 82.5 kDa and a pI of 5.36. The gene locus on Drosophila polytene chromosomes was detected at 64C9 on the left arm of chromosome 3 as a single site. The encoded protein showed a relatively high degree of sequence homology with the RAD2 nucleases, especially XPG. Although the XPG-N- and XPG-I-domains are highly conserved in sequence, locations of the domains are similar to those of FEN-1 and EXO-1, and the molecular weight of the protein is close to that of EXO-1. In vitro, DmGEN showed endonuclease and 3'-5' exonuclease activities with both single-stranded DNA (ssDNA) and double-stranded DNA (dsDNA), but the endonuclease action with dsDNA was quite specific: 5'-3' exonuclease activity was found to occur with nicked DNA, while dsDNA was endonucleolytically cut at 3-4 bp from the 5' end. Homologs are widely found in mammals and higher plants. The data suggest that DmGEN belongs to a new class of RAD2 nuclease.
We have focused attention on functions of Drosophila damaged DNA binding protein 1 (D-DDB1) in Drosophila hematopoiesis and previously reported that its whole body dsRNA over-expression using a GAL4-UAS targeted expression system results in melanotic tumors and complete lethality. Since the lesions appear to arise as a normal and heritable response to abnormal development, forming groups of cells that are recognized by the immune system and encapsulated in melanized cuticle, D-DDB1 appears to be an essential development-associated factor in Drosophila. To probe the possibility that it contributes to hemocyte development, we used a collagen promoter-GAL4 strain to over-express dsRNA of D-DDB1 in Drosophila hemocytes. The D-DDB1 gene silencing caused melanotic tumors and mortality at the end of larval development. Similarly, it interfered with melanization and synthesis of antimicrobial peptides. Transgenic flies with D-DDB1 gene silencing were found to accumulate abnormal large blood cells, reminiscent of human leukemia, suggesting that D-DDB1 has functions in hemocyte development.
Studies on the biochemical properties of very-large-size eukaryotic DNA polymerases have been limited by the difficulty in obtaining sufficient purified forms of each enzyme. Our aim was to determine and elucidate the biochemical properties of one such polymerase, pol zeta (DNA polymerase zeta) from Drosophila melanogaster (Dmpol zeta). Using an REV1 (UV-revertible gene 1) protein-affinity column, we have isolated the enzyme directly from Drosophila embryos. Completely purified Dmpol zeta was found to have a molecular mass of approx. 240 kDa, and to be sensitive to aphidicolin and resistant to ddTTP (2',3'-dideoxythymidine-5-triphosphate) and N-ethylmaleimide. The enzyme has a preference for poly(dA)/oligo(dT)(10:1) as a template primer and has high processivity for DNA synthesis. Moreover, Dmpol zeta showed significantly higher fidelity compared with Rattus norvegicus DNA polymerase, an error-prone DNA polymerase, in an M13 forward mutation assay. The activities of bypassing pyrimidine dimers and (6-4) photoproducts and extending from mismatched primer-template termini in (6-4) photoproduct by Dmpol zeta were not detected. Drosophila REV7 interacted with Dmpol zeta in vitro, but did not influence the DNA synthesis activity of Dmpol zeta. The present study is the first report about characterization of purified pol zeta from multicellular organisms, and the second concerning the characterization of yeast pol zeta.
We found that untenone A and mannzamenone A inhibit mammalian DNA polymerases alpha and beta, and human terminal deoxynucleotidyl transferase (TdT). The syntheses of both compounds and the structure-activity relationships of untenone A derivatives are described.
The aim of this study was to determine the areas involved in episodes of transient global amnesia (TGA) by calculation of cerebral blood flow (CBF) using 3DSRT, fully automated ROI analysis software which we recently developed. Technetium-99m L, L-ethyl cysteinate dimer single-photon emission tomography ((99m)Tc-ECD SPET) was performed during and after TGA attacks on eight patients (four men and four women; mean study interval, 34 days). The SPET images were anatomically standardized using SPM99 followed by quantification of 318 constant ROIs, grouped into 12 segments (callosomarginal, precentral, central, parietal, angular, temporal, posterior cerebral, pericallosal, lenticular nucleus, thalamus, hippocampus and cerebellum), in each hemisphere to calculate segmental CBF (sCBF) as the area-weighted mean value for each of the respective 12 segments based on the regional CBF in each ROI. Correlation of the intra- and post-episodic sCBF of each of the 12 segments of the eight patients was estimated by scatter-plot graphical analysis and Pearson's correlation test with Fisher's Z-transformation. For the control, (99m)Tc-ECD SPET was performed on eight subjects (three men and five women) and repeated within 1 month; the correlation between the first and second sCBF values of each of the 12 segments was evaluated in the same way as for patients with TGA. Excellent reproducibility between the two sCBF values was found in all 12 segments of the control subjects. However, a significant correlation between intra- and post-episodic sCBF was not shown in the thalamus or angular segments of TGA patients. The present study was preliminary, but at least suggested that thalamus and angular regions are closely involved in the symptoms of TGA.
gfLittle is known at present about the biochemical properties of very large-sized Drosophila DNA polymerases. In a previous study, we tried to purify Drosophila pol. catalytic subunit from embryos through seven column chromatographies and study its biochemical properties. However, we failed to characterize it precisely because an insufficient amount of the enzyme was generated. In this report, we describe direct purification from Drosophila embryos to near homogeneity using Drosophila DNA polymerase second subunit (Drosophila pol. 2) protein-conjugated affinity column chromatography and characterization of the enzyme in detail. To our knowledge this is the first demonstration of native DNA polymerase purification with activity using a subunit protein-affinity column. We observed new characteristics of Drosophila pol. catalytic subunit as follows: Drosophila pol. catalytic subunit synthesized DNA processively in the presence of both Mn(2+) and Mg(2+) ions, but Mn(2+) inhibited the 3'-5' proofreading activity, thereby decreasing the fidelity of DNA replication by 50%.
[reaction: see text] Highly selective cross [2 + 2 + 2] cycloaddition of two different monoynes is achieved by using a catalytic amount of [Ir(cod)Cl](2) and ligand. The ligand had a considerable effect on the reaction. When 1,2-bis(diphenylphosphino)ethane was used, two molecules of dimethyl acetylenedicarboxylate (DMAD) reacted with one molecule of a monoyne to give the 2:1 coupling product. When 1,2-bis(dipentafluorophenylphosphino)ethane was used instead of dppe, one molecule of DMAD reacted with two molecules of a monoyne to give the 1:2 coupling product.
Sulphoquinovosyl diacylglycerol (SQDG) was reported as a selective inhibitor of eukaryotic DNA polymerases alpha and beta [Hanashima, Mizushina, Ohta, Yamazaki, Sugawara and Sakaguchi (2000) Jpn. J. Cancer Res. 91, 1073-1083] and an immunosuppressive agent [Matsumoto, Sahara, Fujita, Shimozawa, Takenouchi, Torigoe, Hanashima, Yamazaki, Takahashi, Sugawara et al. (2002) Transplantation 74, 261-267]. The purpose of this paper is to elucidate the biochemical properties of the inhibition more precisely. As expected, SQDG could inhibit the activities of mammalian DNA polymerases such as alpha, delta, eta and kappa in vitro in the range of 2-5 micro M, and beta and lambda in vitro in the range of 20-45 micro M. However, SQDG could inhibit only mammalian DNA polymerases epsilon (pol epsilon) activity at less than 0.04 micro M. SQDG bound more tightly to mammalian pol epsilon than the other mammalian polymerases tested. Moreover, SQDG could inhibit the activities of all the polymerases from animals such as fish and insect, but not of the polymerases from plant and prokaryotes. SQDG should, therefore, be called a mammalian pol epsilon-specific inhibitor or animal polymerase-specific inhibitor. To our knowledge, this represents the first report about an inhibitor specific to mammalian pol epsilon.
The previously reported three-dimensional stereotaxic region of interest (ROI) template (3DSRT-t) for the analysis of anatomically standardized technetium-99m-L,L-ethyl cysteinate dimer (99mTc-ECD) single photon emission computed tomography (SPECT) images was modified for use in a fully automated regional cerebral blood flow (rCBF) quantification software, 3DSRT, incorporating an anatomical standardization engine transplanted from statistical parametric mapping 99 and ROIs for quantification based on 3DSRT-t. Three-dimensional T2-weighted magnetic resonance images of 10 patients with localized infarcted areas were compared with the ROI contour of 3DSRT, and the positions of the central sulcus in the primary sensorimotor area were also estimated. All positions of the 20 lesions were in strict accordance with the ROI delineation of 3DSRT. The central sulcus was identified on at least one side of 210 paired ROIs and in the middle of 192 (91.4%) of these 210 paired ROIs among the 273 paired ROIs of the primary sensorimotor area. The central sulcus was recognized in the middle of more than 71.4% of the ROIs in which the central sulcus was identifiable in the respective 28 slices of the primary sensorimotor area. Fully automated accurate ROI delineation on anatomically standardized images is possible with 3DSRT, which enables objective quantification of rCBF and vascular reserve in only a few minutes using 99mTc-ECD SPECT images obtained by the RVR method.
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