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Kengo Sakaguchi

Publications and source records attributed to Kengo Sakaguchi.

At least 55 records · Page 3Linked to original sources
PubMed2005

Dipeptide alcohol-based inhibitors of eukaryotic DNA polymerase alpha.

We reported previously that a novel dipeptide alcohol, l-homoserylaminoethanol (Hse-Gly-ol), is a selective inhibitor of eukaryotic DNA polymerase epsilon (pol epsilon) [Bioorg. Med. Chem.2004, 12, 957-962]. The discovery suggests that the dipeptide structure could be a chemical frame for a DNA polymerase inhibitor. Therefore, we chemically synthesized 27 different species of dipeptide alcohols, and tested this inhibitory capability. Compound 6 (l-aspartylaminoethanol, Asp-Gly-ol) was found to be the strongest pol alpha inhibitor. Compound 6 did not influence the activities of other replicative DNA polymerases such as delta and epsilon, and had no effect on the activities of prokaryotic DNA polymerases, nor DNA metabolic enzymes such as human immunodeficiency virus type 1 reverse transcriptase, T7 RNA polymerase and bovine deoxyribonuclease I. The inhibitory effect of compound 6 on pol alpha was dose-dependent, and 50% inhibition was observed at a concentration of 33.5 microM. Compound 6-induced inhibition of pol alpha activity was non-competitive with both the DNA template-primer and the dNTP substrate. This is the first report on a water-soluble pol alpha-specific inhibitor, sought for precise biochemical studies of pol alpha. The relationships between the structures of dipeptide alcohols and the inhibition of eukaryotic DNA polymerases are discussed.

Alcohols↗
PubMed2005

Inhibitory effect of conjugated eicosapentaenoic acid on human DNA topoisomerases I and II.

DNA topoisomerases (topos) and DNA polymerases (pols) are involved in many aspects of DNA metabolism such as replication reactions. We reported previously that long chain unsaturated fatty acids such as polyunsaturated fatty acids (PUFA) (i.e., eicosapentaenoic acid (EPA) and docosahexanoic acid (DHA)) inhibited the activities of eukaryotic pols in vitro. In the present study, we found that PUFA also inhibited human topos I and II activities, and the inhibitory effect of conjugated fatty acids converted from EPA and DHA (cEPA and cDHA) on pols and topos was stronger than that of normal EPA and DHA. cEPA and cDHA inhibited the activities of mammalian pols and human topos, but did not affect the activities of plant and prokaryotic pols or other DNA metabolic enzymes tested. cEPA was a stronger inhibitor than cDHA with IC(50) values for mammalian pols and human topos of 11.0-31.8 and 0.5-2.5 microM, respectively. Therefore, the inhibitory effect of cEPA on topos was stronger than that on pols. Preincubation analysis suggested that cEPA directly bound both topos I and II, but did not bind or interact with substrate DNA. This is the first report that conjugated PUFA such as cEPA act as inhibitors of pols and topos. The results support the therapeutic potential of cEPA as a leading anti-cancer compound that poisons pols and topos.

Animals↗
PubMed2005

Applications of zeolite inorganic composites in biotechnology: current state and perspectives.

The purpose of this short review is to introduce applications of inorganic composites, zeolites, in biotechnology. Although inorganic chemistry is generally considered distant from biotechnology, the two could be harmoniously integrated for biopolymer chromatography. New chromatographic carriers have been developed based on principles differing from those underlying conventional chromatography. Some can be used for the purification of proteins according to novel physicochemical principles, according to their isoelectric point (pI), molecular weight and shape. The amount of protein adsorbed is related to the pore size of the composites, which can recognize biomolecules with reference to these three parameters. Proteins adsorbed at their pI have been found to be desorbed at the pI by polyethylene glycol, but not by high ionic medium (NaCl), SDS, non-ionic detergents, ATP or urea. Therefore, inorganic composites synthesized in consideration of pore size and three-dimensional structure are suitable as new chromatographic carriers. Selective fractionation of biomaterials including proteins and nucleic acids should provide useful information regarding whether conjugated proteins in a precipitated state can be separated on net charge and whether cells can be directly fractionated in future.

Adsorption↗
PubMed2005

A method for the molecular imprinting of hemoglobin on silica surfaces using silanes.

A new molecular imprinting technique using covalently immobilized hemoglobin (Hb) is described for creating Hb-specific recognition cavities on silica. Two kinds of organic silane (3-aminopropyltrimethoxysilane: APTMS, and trimethoxypropylsilane: TMPS) were polymerized on a surface of porous silica after the Hb template was covalently immobilized by forming imine bonds, and their influence was analyzed. The results showed that not only the silane amount but also the relative proportions play an important role in protein imprinting. Pore size distribution on Hb imprinted silica was determined by nitrogen adsorption/desorption after removing the template Hb. The Hb-imprinted silica using covalently immobilized Hb (MIPi) as a template proved superior to silica using free Hb (MIPf) regarding displacement of template Hb, and selective re-adsorption as compared with other non-template proteins. The results suggested the capacity for selective adsorption of MIPi to be not only based on the isoelectric point (pI) and protein molecular weight, but also the characteristics of protein recognition cavities imprinted on base silica.

Adsorption↗
PubMed2005

Effects of DNA polymerase inhibitory and antitumor activities of lipase-hydrolyzed glycolipid fractions from spinach.

We succeeded in purifying the major glycolipid fraction in the class of sulfoquinovosyl diacylglycerol, monogalactosyl diacylglycerol and digalactosyl diacylglycerol (DGDG) from a green vegetable, spinach (Spinacia oleracea L.). This glycolipid fraction was an inhibitor of DNA polymerases and a growth inhibitor of NUGC-3 human gastric cancer cells, and, interestingly, the activities were much stronger when the fraction was hydrolyzed by lipase. Glycolipids in the hydrolyzed fraction consisted of sulfoquinovosyl monoacylglycerol (SQMG), monogalactosyl monoacylglycerol (MGMG) and DGDG. In the in vivo antitumor assay using Greene's melanoma, the fraction containing SQMG, MGMG and DGDG showed to be a promising suppressor of solid tumors. Spinach glycolipid fraction might be a potent antitumor compound if directly injected into a tumor-carrying body, and this fraction may be a healthy food material that has antitumor activity.

Animals↗
PubMed2005

Knockdown of LIM15/DMC1 in the mushroom Coprinus cinereus by double-stranded RNA-mediated gene silencing.

The basidiomycete Coprinus cinereus has many advantages as a model organism for studying sexual development and meiosis, but it has been difficult to investigate using reverse-genetics methods, such as gene disruption by homologous recombination. Here, gene repression by dsRNA-mediated gene silencing was tried as an alternative method for reverse-genetics studies. It was shown that transformation of the LIM15/DMC1 dsRNA expression construct (LIM15dsRNA) resulted in genomic insertion of LIM15dsRNA and paucity of the LIM15/DMC1 transcript. First, LIM15dsRNA was transformed into the homothallic strain AmutBmut to generate a homozygote in which both nuclei had a copy of LIM15dsRNA. The LIM15/DMC1-repressed strain showed abnormal homologous chromosome synapsis during meiosis. Basidiospore production was reduced to 16 % by the induction of dsRNA. However, approximately 60 % of basidiospores were viable. Next, a heterozygote was generated in which one nucleus had a copy of LIM15dsRNA. The phenotype was similar to that of the homozygote. These results are not only the first demonstration of dsRNA-mediated gene silencing in a member of the homobasidiomycete fungi, to which 90 % of mushroom species belong, but also the first successful use of a reverse-genetics approach in C. cinereus research.

Adenosine Triphosphatases↗
PubMed2005

Mammalian mitotic centromere-associated kinesin (MCAK): a new molecular target of sulfoquinovosylacylglycerols novel antitumor and immunosuppressive agents.

Sulfoquinovosylacylglycerols (SQAGs), in particular compounds with C18 fatty acid(s) on the glycerol moiety, may be clinically promising antitumor and/or immunosuppressive agents. They were found originally as inhibitors of mammalian DNA polymerases. However, SQAGs can arrest cultured mammalian cells not only at S phase but also at M phase, suggesting they have several molecular targets. A screen for candidate target molecules using a T7 phage display method identified an amino acid sequence. An homology search showed this to be a mammalian mitotic centromere-associated kinesin (MCAK), rather than a DNA polymerase. Analyses showed SQAGs bound to recombinant MCAK with a K(D)=3.1x10(-4) to 6.2x10-5 M. An in vivo microtubule depolymerization assay, using EGFP-full length MCAK fusion constructs, indicated inhibition of the microtubule depolymerization activity of MCAK. From these results, we conclude that clinically promising SQAGs have at least two different molecular targets, DNA polymerases and MCAK. It should be stressed that inhibitors of MCAK have never been reported previously so that there is a major potential for clinical utility.

Amino Acid Sequence↗
PubMed2005

Two types of replication protein A in seed plants.

Replication protein A (RPA), a heterotrimeric protein composed of 70, 32 and 14-kDa subunits, has been shown to be essential for DNA replication, repair, recombination, and transcription. Previously, we found that, in two seed plants, rice and Arabidopsis, there are two different types of RPA70-kDa subunit. Substantial biochemical and genetic characterization of these two subunits, termed OsRPA70a and OsRPA70b or AtRPA70a and AtRPA70b, respectively, is described in this report. Inactivation of AtRPA70a by transfer DNA insertion or RNA interference is lethal, so the complex containing RPA70a may be essential for DNA replication. Transfer DNA insertion and RNAi lines for AtRPA70b are morphologically normal, albeit hypersensitive to certain mutagens, such as UV-B and methyl methanesulfonate, suggesting that RPA70b functions mostly in DNA repair. In two-hybrid, pull-down and coexpression analysis, OsRPA70b was found to interact more selectively than OsRPA70a with OsRPA32. The data suggest that two different types of RPA heterotrimer are present in seed plants, and that there may be additional 32 and 14-kDa subunit homologs that interact with OsRPA70a. Each of the two probable plant RPA complexes may have different roles in DNA metabolism.

Arabidopsis↗
PubMed2005

Sulfoquinovosylmonoacylglycerol inhibitory mode analysis of rat DNA polymerase beta.

We have previously reported that sulfoquinovosylmonoacylglycerol (SQMG) is a potent inhibitor of mammalian DNA polymerases. DNA polymerase beta (pol beta) is one of the most important enzymes protecting the cell against DNA damage by base excision repair. In this study, we characterized the inhibitory action of SQMG against rat pol beta. SQMG competed with both the substrate and the template-primer for binding to pol beta. A gel mobility shift assay and a polymerase activity assay showed that SQMG competed with DNA for a binding site on the N-terminal 8-kDa domain of pol beta, subsequently inhibiting its catalytic activity. Fragments of SQMG such as sulfoquinovosylglycerol (SQG) and fatty acid (myristoleic acid, MA) weakly inhibited pol beta activity and the inhibitory effect of a mixture of SQG and MA was stronger than that of SQG or MA. To characterize this inhibition more precisely, we attempted to identify the interaction interface between SQMG and the 8-kDa domain by NMR chemical shift mapping. Firstly, we determined the binding site on a fragment of SQMG, the SQG moiety. We observed chemical shift changes primarily at two sites, the residues comprising the C-terminus of helix-1 and the N-terminus of helix-2, and residues in helix-4. Finally, based on our present results and our previously reported study of the interaction interface of fatty acids, we constructed two three-dimensional models of a complex between the 8-kDa domain and SQMG and evaluated them by the mutational analysis. The models show a SQMG interaction interface that is consistent with the data.

Animals↗
PubMed2005

Cell adsorption and selective desorption for separation of microbial cells by using chitosan-immobilized silica.

Cell adsorption and selective desorption for separation of microbial cells were conducted by using chitosan-immobilized silica (CIS). When chitosan was immobilized onto silica surfaces with glutaraldehyde, bacterial cells adsorbed well and retained viability. Testing of the adsorption and desorption ability of CIS using various microbes such as Escherichia coli, Aeromonas hydrophila, Pseudomonas aeruginosa, Bacillus subtilis, Micrococcus luteus, Staphylococcus aureus, Staphylococcus epidermidis, Lactobacillus casei, Streptococcus mutans, Streptococcus sobrinus, Streptococcus salivarius, Saccharomyces cerevisiae, Saccharomyces ludwigii, and Schizosaccharomyces pombe revealed that most microbes could be adsorbed and selectively desorbed under different conditions. In particular, recovery was improved when L-cysteine was added. A mixture of two bacterial strains adsorbed onto CIS could also be successfully separated by use of specific solutions for each strain. Most of the desorbed cells were alive. Thus, quantitative and selective fractionation of cells is readily achievable by employing chitosan, a known antibacterial material.

Adsorption↗
PubMed2005

Sulfo-quinovosyl-acyl-glycerol (SQAG), a eukaryotic DNA polymerase inhibitor and anti-cancer agent.

It was found that a class of sulfolipids known as sulfo-quinovosyl-acyl-glycerols (SQAGs) from ferns and algae are potent inhibitors of eukaryotic DNA polymerase alpha and beta and effective anti-neoplastic agents. In developing a procedure for the chemical synthesis of sulfolipids, many derivatives and stereoisomers of SQAGs have been obtained including sulfo-quinovosyl-monoacyl-glycerols (SQMGs) and sulfo-quinovosyl-diacyl-glycerols (SQDGs). This review describes studies on the structure-function relationship between synthetic SQAGs and DNA polymerase alpha and beta, and the relationship to cytotoxic activity. The major action was probably dependent on the fatty acid effect, which was reported previously, although each of the SQAGs was a much stronger inhibitor than just the fatty acid present in the SQAGs. The inhibitory effect could be influenced by the chain size of fatty acids in the SQAGs. The sulfonyl group in quinovose was also needed to inhibit the enzymes. Lineweaver-Burk plots of SQAGs indicated that DNA polymerase alpha was non-competitively inhibited, but the SQAGs were effective as antagonists of both the template-primer DNA-binding and the nucleotide substrate-binding of DNA polymerase beta. Based on these results, the molecular actions of SQAGs and drug design strategies for developing new anti-neoplastic agents were discussed.

Animals↗
PubMed2005

Structural analysis of epolactaene derivatives as DNA polymerase inhibitors and anti-inflammatory compounds.

Epolactaene (compound 1), a neuritogenic compound found in human neuroblastoma cells, was found to show anti-inflammatory activity in vivo in this study. DNA polymerases and DNA topoisomerase II (topo II) were some of the major molecular targets of compound 1. Since the agent seems to be a potential pharmaceutical medicine, we synthesized derivatives chemically and obtained seven compounds, 1 to 7 to screen clinically more efficient epolactaene derivatives. A comparison of its structural derivatives revealed that the long alkyl side chain seemed to have an important role in the inhibitory effect. Notably, C18-alkyl chain conjugated epolactaene (compound 5) was the strongest inhibitor of DNA polymerase alpha, beta, lambda (pol alpha, beta, lambda) and topo II, with IC50 values of 13, 135, 4.4 and 5 microM, respectively, and 500 microg of compound 5 caused a marked reduction in TPA (12-O-tetradecanoylphorbol-13-acetate)-induced inflammation (inhibitory effect, 65.0%). Compound 5 did not influence the activities of plant or prokaryotic DNA polymerases, or of other DNA metabolic enzymes such as telomerase, RNA polymerase and deoxyribonuclease I. Based on these results, the relationship among the three-dimensional structure of epolactaene derivatives and the inhibition of polymerases and topo II, and anti-inflammation is discussed.

Animals↗
PubMed2004

Design of vesicles of 1,2-di-O-acyl-3-O-(beta-D-sulfoquinovosyl)-glyceride bearing two stearic acids (beta-SQDG-C18), a novel immunosuppressive drug.

The immunosuppressive effects of synthetic sulfo-glycolipids in the class of sulfoquinovosyl-diacylglycerols (SQDG), including stereoisomers, were interesting in development of a promising clinical drug. Especially, 1,2-di-O-stearoyl-3-O-(6-deoxy-6-sulfo-beta-D-glucopyranosyl)-sn-glycerol (beta-SQDG-C18) was thought to be a valuable candidate because of the preliminary observations of its high inhibitory activities in spite of low toxicities. The problem of using this material is to find an applicable way avoiding its low solubility in water. The vesicle formation of beta-SQDG-C18 is advantageous to i.v. administration in its chemico-structural character. With preparation in water, beta-SQDG-C18 was hard to form vesicles, because its hydrophilicity was strong. We examined the suitable parameter of the vesicle forming condition. It was possible to take a balance between the hydrophilicity and the hydrophobicity of the beta-SQDG-C18 molecule to be optimized to form vesicles in 150 mM PBS. In addition, we demonstrated the strong immunosuppressive activity of beta-SQDG-C18 vesicles. This is the first report of the preparation method of beta-SQDG-C18 vesicles, which should facilitate in vitro and in vivo application.

Drug Stability↗
PubMed2004

DmGEN, a novel RAD2 family endo-exonuclease from Drosophila melanogaster.

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.

Animals↗
PubMed2004

Monolayer membranes and bilayer vesicles characterized by alpha- and beta-anomer of sulfoquinovosyldiacyglycerol (SQDG).

Physicochemical properties of 1,2-di-O-stearoyl-3-O-(6-deoxy-6-sulfo-alpha-D-glucopyranosyl)-sn-glycerol (alpha-SQDG-C(18:0)) and 1,2-di-O-stearoyl-3-O-(6-deoxy-6-sulfo-beta-D-glucopyranosyl)-sn-glycerol (beta-SQDG-C(18:0)) in monolayer and bilayer membranes were examined. Surface pressure measurements in monolayer membranes indicated the molecular area of beta-SQDG-C(18:0) to be slightly smaller than that of alpha-SQDG-C(18:0). In bilayer membranes, the phase transition temperature and the enthalpy of beta-SQDG-C(18:0) were higher than those of alpha-SQDG-C(18:0), while the trapping efficiency of beta-SQDG-C(18:0) vesicles was lower. The results suggested tighter packing with beta-SQDG-C(18:0) than alpha-SQDG-C(18:0), due to differences in the head group stereochemistry. High-performance liquid chromatography-electrospray ionization ion trap mass spectrometry (HPLC-ESI-MS) data and computational modeling studies provided supporting evidence for morphological differences. In both monolayer and bilayer membranes, the affinity of beta-SQDG-C(18:0) with cholesterol was greater than that of alpha-SQDG-C(18:0), again due to the differences in head group properties. Turbidity measurement and microscopic examination of alpha- and beta-SQDG-C(18:0)/cholesterol mixtures confirmed formation of large vesicles. The addition of cholesterol to SQDG-C(18:0) optimized membrane formation and stabilized its structure.

Calorimetry, Differential Scanning↗
PubMed2004

Damaged DNA binding protein 1 in Drosophila defense reactions.

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.

Animals↗
PubMed2004

Precise structural elucidation of dehydroaltenusin, a specific inhibitor of mammalian DNA polymerase alpha.

The X-ray crystal structure of dehydroaltenusin, a specific inhibitor of mammalian DNA polymerase alpha, has previously been reported. We show that dehydroaltenusin exists in an equilibrium mixture of two tautomers possessing gamma-lactone or delta-lactone in polar solvents by NMR experiments. Acetylation of dehydroaltenusin afforded two types of diacetates and two types of monoacetate, possessing gamma-lactone or delta-lactone, respectively. The inhibitory activities of these acetate derivatives against DNA polymerase alpha were all much weaker than that of dehydroaltenusin.

Acetylation↗