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Hiroshi Ochiai - One of the best experts on this subject based on the ideXlab platform.
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promoter analysis of the membrane protein gp64 gene of the Cellular Slime Mold polysphondylium pallidum
Biochimica et Biophysica Acta, 1999Co-Authors: Naohisa Takaoka, Tamao Saito, Masashi Fukuzawa, Takehisa Sakaitani, Hiroshi OchiaiAbstract:Abstract We cloned a genomic fragment of the membrane protein gp64 gene of the Cellular Slime Mold Polysphondylium pallidum by inverse PCR. Primer extension analysis identified a major transcription start site 65 bp upstream of the translation start codon. The promoter region of the gp64 gene contains sequences homologous to a TATA box at position −47 to −37 and to an initiator (Inr, PyPyCAPyPyPyPy) at position −3 to +5 from the transcription start site. Successively truncated segments of the promoter were tested for their ability to drive expression of the β-galactosidase reporter gene in transformed cells; also the difference in activity between growth conditions was compared. The results indicated that there are two positive vegetative regulatory elements extending between −187 and −62 bp from the transcription start site of the gp64 promoter; also their activity was two to three times higher in the cells grown with bacteria in shaken suspension than in the cells grown in an axenic medium.
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Identification of Delta5-fatty acid desaturase from the Cellular Slime Mold dictyostelium discoideum.
European journal of biochemistry, 1999Co-Authors: Tamao Saito, Hiroshi OchiaiAbstract:cDNA fragments putatively encoding amino acid sequences characteristic of the fatty acid desaturase were obtained using expressed sequence tag (EST) information of the Dictyostelium cDNA project. Using this sequence, we have determined the cDNA sequence and genomic sequence of a desaturase. The cloned cDNA is 1489 nucleotides long and the deduced amino acid sequence comprised 464 amino acid residues containing an N-terminal cytochrome b5 domain. The whole sequence was 38.6% identical to the initially identified Delta5-desaturase of Mortierella alpina. We have confirmed its function as Delta5-desaturase by over expression mutation in D. discoideum and also the gain of function mutation in the yeast Saccharomyces cerevisiae. Analysis of the lipids from transformed D. discoideum and yeast demonstrated the accumulation of Delta5-desaturated products. This is the first report concering fatty acid desaturase in Cellular Slime Molds.
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fatty acid composition of the Cellular Slime Mold polysphondylium pallidum
Lipids, 1998Co-Authors: Tamao Saito, Hiroshi OchiaiAbstract:The Cellular Slime Mold Polysphondylium pallidum was grown upon Escherichia coli B/r, and the fatty acid compositions of total lipids obtained from vegetative amebae and aggregation-competent cells were compared. Fatty acids isolated from vegetative cells included C-17 and C-19 cyclopropane fatty acids and also straight-chain, saturated fatty acids. The cyclopropane fatty acids were derived from the ingested bacteria. Development of amebae to aggregation-competent cells was accompanied by a substantial decrease in saturated cyclopropane fatty acids and a concomitant increase in unsaturated fatty acids and unsaturated cyclopropane fatty acids, mostly as 18∶3 (5,9,12). We report here the fatty acid composition and identify the occurrence of Δ5 desaturation of cyclopropane fatty acids, namely, 9,10-methylene 5-hexadecenoic acid and 11,12-methylene 5-octadecenoic acid. These fatty acids have not been reported previously in the related species Dictyostelium discoideum, which also feeds on E. coli B/r and has Δ5-desaturation activity.
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molecular cloning and the cooh terminal processing of gp64 a putative cell cell adhesion protein of the Cellular Slime Mold polysphondylium pallidum
Journal of Biological Chemistry, 1994Co-Authors: R I Manabe, T Kumazaki, Takehisa Sakaitani, Takeshi Saito, N Nakata, Hiroshi OchiaiAbstract:The Cellular Slime Mold Polysphondylium pallidum expresses a cell surface glycoprotein (referred to as gp64), which seems to be implicated in cell-cell adhesion. We identified a near full-length gp64 cDNA (1,104 base) upon screening a P. pallidum lambda gt11 library with a monoclonal antibody. The open reading frame encodes a protein of 320 amino acids with a molecular mass of 32,752 Da; the protein includes hydrophobic segments at both a NH2- and a COOH-terminal ends. By an Edman degradation analysis of S-pyridylethylated gp64 and its COOH-terminal peptide, it was found that the NH2- and COOH-terminal segments are both removed from the precursor protein of gp64. The COOH-terminal segment was isolated from a lysyl endopeptidase digest of gp64 by an affinity method. The COOH-terminal segment was identified at positions 266-279 in the primary sequence deduced from the cDNA sequence. The mature gp64 consisted of 279 amino acid residues and extremely rich in Cys residues (36 Cys/279 amino acids = 12.9%). Although there was already maximal accumulation of gp64 mRNA in vegetative cells, the protein reached a maximal level during aggregation stage, decreased, and then leveled off through the developmental cycle.
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a purification method and n glycosylation sites of a 36 cysteine containing putative cell cell adhesion glycoprotein gp64 of the Cellular Slime Mold polysphondylium pallidum
FEBS Journal, 1993Co-Authors: Tamao Saito, Takashi Kumazaki, Hiroshi OchiaiAbstract:A 64-kDa membrane-bound glycoprotein (gp64) of the Cellular Slime Mold Polysphondylium pallidum, is a putative cell/cell adhesion protein identified by adhesion-blocking antibody fragments (Fab). gp64 is expressed on the cell surface of growth-phase cells and seems to mediate cell/cell adhesion. This paper describes an improved purification method based on the lipophilic nature of this protein. A critical step in the purification method is to collect an insoluble top layer appearing during ammonium sulfate precipitation. The sequence of cDNA encoding gp64 and its deduced amino acid sequence have been determined previously. Based on cDNA sequence data, the structure of gp64 protein was analyzed: almost all amino acid compositions and partial amino acid sequences of lysylendopeptidase-digested peptides of gp64 were determined by protein analysis; all six asparagine-linked glycosylation sites (Asn-Xaa-Ser/Thr) in fact contain carbohydrates, and all 36 cysteine residues were involved in forming disulfide bridges. From these data, gp64 seems to be a unique protein among cell/cell adhesion proteins.
Tamao Saito - One of the best experts on this subject based on the ideXlab platform.
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Chemical compounds from Dictyostelium discoideum repel a plant-parasitic nematode and can protect roots
2018Co-Authors: Yumiko F. Saito, Saki H. Miyazaki, Derek G. Bartlem, Yukiko Nagamatsu, Tamao SaitoAbstract:Slime Mold species in the genus Dictyostelium are considered to have a close relationship with non-parasitic nematodes; they are sympatric in soils and can exhibit interspecific competition for food. We investigated whether this relationship extends to a plant-parasitic nematode that is active in the rhizosphere and has broad host specificity, damaging crops worldwide. Using a novel assay to examine the interaction between the Cellular Slime Mold, Dictyostelium discoideum, and the plant-parasitic nematodes, Meloidogyne spp., we found that Cellular Slime Molds can repel plant parasitic nematodes. Specifically, the repulsion activity was in response to chemical compounds released by Cellular Slime Mold fruiting bodies. Under laboratory conditions, these soluble chemical extracts from fruiting bodies of D. discoideum showed repulsion activity strong enough to protect plant roots. The fruiting body cell extracts repelled but were not toxic to the plant-parasitic nematodes.
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Stalk cell differentiation without polyketides in the Cellular Slime Mold
Bioscience biotechnology and biochemistry, 2016Co-Authors: Yukie G. Sato, Teresa Suárez, Tamao SaitoAbstract:Polyketides induce prestalk cell differentiation in Dictyostelium. In the double-knockout mutant of the SteelyA and B polyketide synthases, most of the pstA cells-the major part of the prestalk cells-are lost, and we show by whole mount in situ hybridization that expression of prestalk genes is also reduced. Treatment of the double-knockout mutant with the PKS inhibitor cerulenin gave a further reduction, but some pstA cells still remained in the tip region, suggesting the existence of a polyketide-independent subtype of pstA cells. The double-knockout mutant and cerulenin-treated parental Ax2 cells form fruiting bodies with fragile, single-cell layered stalks after cerulenin treatment. Our results indicate that most pstA cells are induced by polyketides, but the pstA cells at the very tip of the slug are induced in some other way. In addition, a fruiting body with a single-cell layered, vacuolated stalk can form without polyketides.
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promoter analysis of the membrane protein gp64 gene of the Cellular Slime Mold polysphondylium pallidum
Biochimica et Biophysica Acta, 1999Co-Authors: Naohisa Takaoka, Tamao Saito, Masashi Fukuzawa, Takehisa Sakaitani, Hiroshi OchiaiAbstract:Abstract We cloned a genomic fragment of the membrane protein gp64 gene of the Cellular Slime Mold Polysphondylium pallidum by inverse PCR. Primer extension analysis identified a major transcription start site 65 bp upstream of the translation start codon. The promoter region of the gp64 gene contains sequences homologous to a TATA box at position −47 to −37 and to an initiator (Inr, PyPyCAPyPyPyPy) at position −3 to +5 from the transcription start site. Successively truncated segments of the promoter were tested for their ability to drive expression of the β-galactosidase reporter gene in transformed cells; also the difference in activity between growth conditions was compared. The results indicated that there are two positive vegetative regulatory elements extending between −187 and −62 bp from the transcription start site of the gp64 promoter; also their activity was two to three times higher in the cells grown with bacteria in shaken suspension than in the cells grown in an axenic medium.
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Identification of Delta5-fatty acid desaturase from the Cellular Slime Mold dictyostelium discoideum.
European journal of biochemistry, 1999Co-Authors: Tamao Saito, Hiroshi OchiaiAbstract:cDNA fragments putatively encoding amino acid sequences characteristic of the fatty acid desaturase were obtained using expressed sequence tag (EST) information of the Dictyostelium cDNA project. Using this sequence, we have determined the cDNA sequence and genomic sequence of a desaturase. The cloned cDNA is 1489 nucleotides long and the deduced amino acid sequence comprised 464 amino acid residues containing an N-terminal cytochrome b5 domain. The whole sequence was 38.6% identical to the initially identified Delta5-desaturase of Mortierella alpina. We have confirmed its function as Delta5-desaturase by over expression mutation in D. discoideum and also the gain of function mutation in the yeast Saccharomyces cerevisiae. Analysis of the lipids from transformed D. discoideum and yeast demonstrated the accumulation of Delta5-desaturated products. This is the first report concering fatty acid desaturase in Cellular Slime Molds.
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fatty acid composition of the Cellular Slime Mold polysphondylium pallidum
Lipids, 1998Co-Authors: Tamao Saito, Hiroshi OchiaiAbstract:The Cellular Slime Mold Polysphondylium pallidum was grown upon Escherichia coli B/r, and the fatty acid compositions of total lipids obtained from vegetative amebae and aggregation-competent cells were compared. Fatty acids isolated from vegetative cells included C-17 and C-19 cyclopropane fatty acids and also straight-chain, saturated fatty acids. The cyclopropane fatty acids were derived from the ingested bacteria. Development of amebae to aggregation-competent cells was accompanied by a substantial decrease in saturated cyclopropane fatty acids and a concomitant increase in unsaturated fatty acids and unsaturated cyclopropane fatty acids, mostly as 18∶3 (5,9,12). We report here the fatty acid composition and identify the occurrence of Δ5 desaturation of cyclopropane fatty acids, namely, 9,10-methylene 5-hexadecenoic acid and 11,12-methylene 5-octadecenoic acid. These fatty acids have not been reported previously in the related species Dictyostelium discoideum, which also feeds on E. coli B/r and has Δ5-desaturation activity.
Yoshiteru Oshima - One of the best experts on this subject based on the ideXlab platform.
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production of novel bispyrone metabolites in the Cellular Slime Mold dictyostelium giganteum induced by zinc ii ion
Tetrahedron, 2017Co-Authors: Van Hai Nguyen, Haruhisa Kikuchi, Yuzuru Kubohara, Hiraku Sasaki, Kyoichi Iizumi, Yoshiteru OshimaAbstract:Abstract In this study, Zn2+ induced the production of two new bispyrone-type metabolites, dictyobispyrone B and E, in the Cellular Slime Mold Dictyostelium giganteum. Their structures were proposed on the basis of spectroscopic analysis and confirmed by chemical synthesis. They possess a novel α,α-bispyrone skeleton modified with long carbon chains. Both could be formed from two different polyketide chains through a novel biosynthetic pathway. Our results indicate that cultivation of Cellular Slime Molds in the presence of Zn2+ is a useful technique for discovering other structurally unique compounds.
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novel chlorinated dibenzofurans isolated from the Cellular Slime Mold polysphondylium filamentosum and their biological activities
Bioorganic & Medicinal Chemistry, 2013Co-Authors: Haruhisa Kikuchi, Yuzuru Kubohara, Van Hai Nguyen, Yasuhiro Katou, Yoshiteru OshimaAbstract:Cellular Slime Molds are expected to have the huge potential for producing secondary metabolites including polyketides, and we have studied the diversity of secondary metabolites of Cellular Slime Molds for their potential utilization as new biological resources for natural product chemistry. From the methanol extract of fruiting bodies of Polysphondylium filamentosum, we obtained new chlorinated benzofurans Pf-1 (4) and Pf-2 (5) which display multiple biological activities; these include stalk cell differentiation-inducing activity in the well-studied Cellular Slime Mold, Dictyostelium discoideum, and inhibitory activities on cell proliferation in mammalian cells and gene expression in Drosophila melanogaster.
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anti leukemic activities of dictyostelium secondary metabolites a novel aromatic metabolite 4 methyl 5 n pentylbenzene 1 3 diol isolated from dictyostelium mucoroides suppresses cell growth in human leukemia k562 and hl 60 cells
Life Sciences, 2006Co-Authors: Haruhisa Kikuchi, Yoshiteru Oshima, Aya Ichimura, Naomi Gokan, Aiko Hasegawa, Kohei Hosaka, Yuzuru KuboharaAbstract:Abstract It has previously been shown that DIF-1, a differentiation-inducing factor of the Cellular Slime Mold Dictyostelium discoideum, possesses antitumor activities in mammalian tumor cells and that neuronal differentiation of PC12 cells can be induced with furanodictines (FDs), aminosugar analogs found in D. discoideum , or dictyoglucosamines (DGs), N -acetyl glucosamine derivatives (DG-A from D. purpureum and DG-B from D. discoideum ). Thus, Cellular Slime Molds are attractive natural resources that may provide valuable lead compounds to be utilized in the field of pharmacology and medicine. In this study, we have isolated a novel aromatic compound, 4-methyl-5- n -pentylbenzene-1,3-diol (MPBD), from fruiting bodies of the Cellular Slime Mold D. mucoroides and assessed the in vitro antiproliferative activities of MPBD, FDs, and DGs in human leukemia K562 and HL-60 cells. MPBD at 20–80 μM dose-dependently suppressed cell growth in both K562 and HL-60 cells. While FDs at 10–80 μM did not affect cell growth, DGs at 10–40 μM dose-dependently suppressed cell growth in the cells. Although we failed to find the roles of FDs and DGs in the original organisms, MPBD at 5–20 μM was found to promote stalk cell formation in D. discoideum . The present results indicate that MPBD, DGs or their derivatives may have therapeutic potential in the treatment of cancer and confirm our expectations regarding Cellular Slime Molds as drug resources.
T Kumazaki - One of the best experts on this subject based on the ideXlab platform.
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Assignment of disulfide bonds in gp64, a putative cell-cell adhesion protein of Polysphondylium pallidum. Presence of Sushi domains in the Cellular Slime Mold protein.
The Journal of biological chemistry, 1994Co-Authors: Tetsuya Saito, T Kumazaki, H OchiaiAbstract:Abstract The 64-kDa membrane-bound glycoprotein of the Cellular Slime Mold Polysphondylium pallidum (referred to as gp64), seems to be implicated in cell-cell adhesion. Previously we have isolated a full-length gp64 cDNA, determined its nucleotide sequence, and found that all cysteine residues in the protein are involved in the formation of disulfide bonds. The disulfide arrangement of the 36 cysteines in gp64 was established by analysis of proteolytically cleaved protein and sequence analysis of cystine-containing fragments. Since gp64 has 36 Cys residues, 18 disulfide bonds must exist and the positions of 15 of them were determined. The 15 disulfide bonds in gp64 constitute five characteristic, so-called Sushi domains. In a Sushi domain, the first Cys in a sequence is connected to the third one and the second Cys to the fourth one. This is the first report describing the presence of Sushi domains in a Cellular Slime Mold protein. From these data, gp64 appears to be distinct from all other previously described cell-adhesion proteins.
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molecular cloning and the cooh terminal processing of gp64 a putative cell cell adhesion protein of the Cellular Slime Mold polysphondylium pallidum
Journal of Biological Chemistry, 1994Co-Authors: R I Manabe, T Kumazaki, Takehisa Sakaitani, Takeshi Saito, N Nakata, Hiroshi OchiaiAbstract:The Cellular Slime Mold Polysphondylium pallidum expresses a cell surface glycoprotein (referred to as gp64), which seems to be implicated in cell-cell adhesion. We identified a near full-length gp64 cDNA (1,104 base) upon screening a P. pallidum lambda gt11 library with a monoclonal antibody. The open reading frame encodes a protein of 320 amino acids with a molecular mass of 32,752 Da; the protein includes hydrophobic segments at both a NH2- and a COOH-terminal ends. By an Edman degradation analysis of S-pyridylethylated gp64 and its COOH-terminal peptide, it was found that the NH2- and COOH-terminal segments are both removed from the precursor protein of gp64. The COOH-terminal segment was isolated from a lysyl endopeptidase digest of gp64 by an affinity method. The COOH-terminal segment was identified at positions 266-279 in the primary sequence deduced from the cDNA sequence. The mature gp64 consisted of 279 amino acid residues and extremely rich in Cys residues (36 Cys/279 amino acids = 12.9%). Although there was already maximal accumulation of gp64 mRNA in vegetative cells, the protein reached a maximal level during aggregation stage, decreased, and then leveled off through the developmental cycle.
Hiro Yasukawa - One of the best experts on this subject based on the ideXlab platform.
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developmental and spatial expression of sir2 genes in the Cellular Slime Mold dictyostelium discoideum
Microbes and Environments, 2008Co-Authors: Takahiro Katayama, Hiro YasukawaAbstract:The Cellular Slime Mold Dictyostelium discoideum grows as uniCellular free-living amoebae in the presence of nutrients. Upon starvation, the amoebae aggregate and form multiCellular structures that each consist of a stalk and spores. D. discoideum encodes at least four proteins (Sir2A, Sir2B, Sir2C, and Sir2D) homologous to human SIRT. RT-PCR and WISH analyses showed that the genes for Sir2A, Sir2C, and Sir2D were expressed at high levels in growing cells but at decreased levels in developing cells, whereas the gene encoding Sir2B was expressed in the prestalk-cell region in the developmental phase.
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a homolog of escherichia coli reca in mitochondria of the Cellular Slime Mold dictyostelium discoideum
DNA Repair, 2004Co-Authors: Yasuna Hasegawa, Masayuki Wakabayashi, Shogo Nakamura, Kenichi Kodaira, Hiroaki Shinohara, Hiro YasukawaAbstract:The Cellular Slime Mold Dictyostelium discoideum expresses a gene encoding a 452-amino-acid polypeptide that is 47% identical to Escherichia coli RecA. A recA-deficient E. coli, JE6651, was transformed by pYSN1, which was designed to express the truncated form of the D. discoideum gene, and used in suppression assays. The viability of the transformant, JE6651(pYSN1), increased following UV irradiation or mitomycin C treatment. Phage lambda (red(-) gam(-)), which required RecA activity for DNA packaging, formed plaques on a lawn of JE6651(pYSN1). These results indicate that the gene product has a DNA recombination activity. Fluorescence of D. discoideum protein fused with GFP was detected in mitochondria. The gene disruption mutant was hypersensitive to UV-light (254nm), mitomycin C and H(2)O(2), indicating that D. discoideum recA is important for survival following exposure to DNA damaging agents.
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Identification of a penicillin-sensitive carboxypeptidase in the Cellular Slime Mold Dictyostelium discoideum.
Biological & pharmaceutical bulletin, 2003Co-Authors: Hiro Yasukawa, Toshihiro Kuroita, Kentaro Tamura, Kazuo YamaguchiAbstract:Penicillin binding proteins (PBPs) are penicillin-sensitive DD-peptidases catalyzing the terminal stages of bacterial cell wall assembly. We identified a Dictyostelium discoideum gene that encodes a protein of 522 amino acids showing similarity to Escherichia coli PBP4. The D. discoideum protein conserves three consensus sequences (SXXK, SXN and KTG) that are responsible for the catalytic activities of PBPs. The gene product prepared in the cell-free translation system showed carboxypeptidase activity but the activity was not detected in the presence of penicillin G. These results demonstrate that the D. discoideum gene encodes a eukaryotic form of penicillin-sensitive carboxypeptidase.