論文 - 田村　悦臣

Inhibition of cell growth by bafilomycin A1, a selective inhibitor of vacuolar H+-ATPase.

S. Ohkuma*, S. Shimizu, M. Noto, Y. Sai, K. Kinoshita and H. Tamura

In Vitro Cell Dev Biol 29A （ 11 ） 862 - 866 1993年11月

研究論文（学術雑誌）, 共著, 査読有り

Inhibition of cell growth by bafilomycin A<inf>1</inf>, a selective inhibitor of vacuolar H⁺-ATPase

Ohkuma S., Shimizu S., Noto M., Sai Y., Kinoshita K., Tamura H.

In Vitro Cellular &amp; Developmental Biology - Animal: Journal of the Society for In Vitro Biology （In Vitro Cellular &amp; Developmental Biology - Animal: Journal of the Society for In Vitro Biology）  29 （ 11 ） 862 - 866 1993年11月

研究論文（学術雑誌）,  ISSN  10712690

　概要を見る

Bafilomycin A1, a potent selective inhibitor of vacuolar H+-ATPase, inhibited the growth of a variety of cultured cells dose-dependently, including golden hamster embryo and NIH-3T3 fibroblasts, whether or not they were transformed, and PC12 and HeLa cells. The concentration of bafilomycin A1 for 50% inhibition of cell growth ranged from 10 to 50 n M. The dose response was nearly parallel with that of the bafilomycin A1-induced lysosomal pH increase. The degree of pH increase for growth inhibition produced by bafilomycin A1 was similar to that produced by NH4Cl in which little difference was recognized in effect among cell types. © 1993, Tissue Culture Association. All rights reserved.

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Purification and cDNA molecular cloning of bovine liver 5'-nucleotidase, a GPI-anchored protein, and its expression in COS cells.

田村 悦臣

J Biochem 113 （ 3 ） 607 - 613 1993年05月

研究論文（学術雑誌）, 共著, 査読有り

Mass production of sphingomyelinase of Bacillus cereus by a protein-hyperproducing strain, Bacillus brevis 47 and its purification.

H. Tamura*, K. Tameishi, H. Yamagata, S. Udaka, T. Kobayashi, M. Tomita and H. Ikezawa

J Biochem 112 （ 4 ） 488 - 491 1992年10月

研究論文（学術雑誌）, 共著, 査読有り

Mass production of sphingomyelinase of Bacillus cereus by a protein-hyperproducing strain, Bacillus brevis 47, and its purification

Tamura H., Tameishi K., Yamagata H., Udaka S., Kobayashi T., Tomita M., Ikezawa H.

Journal of Biochemistry （Journal of Biochemistry）  112 （ 4 ） 488 - 491 1992年10月

研究論文（学術雑誌）,  ISSN  0021924X

　概要を見る

Sphingomyelinase (sphingomyelin cholinephosphohydrolase) [EC 3.1.4.12] of Bacillus cereus was overproduced in a protein-hyperproducing strain, B. brevis 47, by cloning the gene into an expression vector pNU211, which has been developed to express a foreign gene utilizing a promoter and a signal sequence of an outer cell wall protein gene. From 1 liter of culture, about 10 mg of protein was purified to near-homogeneity by two steps of column chromatography; this is almost 500 times higher production compared to the conventional preparation from the original strain, B. cereus IAM 1208. The N-terminal amino acid sequence of the secreted enzyme was identical to that of the authentic enzyme, indicating that the signal sequence for secretion of B. cereus was processed properly in B. brevis 47. © 1992 BY THE JOURNAL OF BIOCHEMISTRY.

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Molecular cloning of a rat liver cDNA encoding the 16KDa subunit of vacuolar H+-ATPase; Organella and tissue distribution of 16 KDa proteolipids.

J. Nezu, K. Motojima, H. Tamura and S. Ohkuma*

J Biochem 112 （ 2 ） 212 - 219 1992年08月

研究論文（学術雑誌）, 共著, 査読有り

Molecular cloning of a rat liver cDNA encoding the 16 kDa subunit of vacuolar H⁺-ATPases: Organellar and tissue distribution of 16 kDa proteolipids

Nezu J., Motojima K., Tamura H., Ohkuma S.

Journal of Biochemistry （Journal of Biochemistry）  112 （ 2 ） 212 - 219 1992年08月

研究論文（学術雑誌）,  ISSN  0021924X

　概要を見る

A cDNA (T3-L) encoding the 16 kDa subunit of vacuolar H+-ATPase was cloned from a cDNA library of rat liver. A polypeptide of 156 amino acids with a molecular mass of 15,807 Da (pI=9.5) having four hydrophobic stretches was predicted. T3-L polypeptide was 92% and 100% identical with the 16 kDa proteolipid of bovine chromaffin granule and that of mouse, respectively. Antisera raised against the NH2-terminal of the T3-L polypeptide reacted positively with the membrane ghosts of rat liver tritosomes and the partially purified H+-ATPase thereof. Western blotting of subcellular fractions with the antisera showed high abundance of 16 kDa protein in the lysosomes, although a significant amount was also detected in the Golgi apparatus. Western blotting of rat tissues revealed high levels of 16 kDa proteolipid in the brain and the kidney. Northern blots with T3-L similarly showed considerably high expression of T3-L mRNA in the brain and the kidney. Southern hybridization of rat genomic DNA with T3-L showed at most three distinct bands, regardless of the stringency of hybridization and whether hybridization was performed with its subfragments. This suggests the possibility of multiple (at least three) homologous/identical genes encoding 16 kDa proteolipid. The possible presence and significance of isoforms of 16 kDa proteolipid in rats are discussed. © 1992 THE JOURNAL OF BIOCHEMISTRY.

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Induction of neurite outgrowth of PC12 cells by an inhibitor of vacuolar H+-ATPase, bafilomycin A1.

H. Tamura* and S. Ohkuma

FEBS Lett 294 （ 1-2 ） 51 - 55 1991年12月

研究論文（学術雑誌）, 共著, 査読有り

Induction of neurite outgrowth of PC12 cells by an inhibitor of vacuolar H⁺-ATPase, bafilomycin A<inf>1</inf>

Tamura H., Ohkuma S.

FEBS Letters （FEBS Letters）  294 （ 1-2 ） 51 - 55 1991年12月

研究論文（学術雑誌）,  ISSN  00145793

　概要を見る

Bafilomycin A1, a selective inhibitor of vacuolar H+-ATPase, induced neurite outgrowth of PC12 cells dose- and time-dependently: more than 50% of the cells extended neurite-like spikes after 24 h treatment with 100 nM bafilomycin A1. Its dose-response ran roughly parallel to that of a bafilomycin A1-induced lysosomal pH increase. It was inhibited by LiCl, an inhibitor of the phosphorylation of microtubule-associated proteins and, like nerve growth factor (NGF)-induced neurite outgrowth, it was also inhibited by cycloheximide and actinomycin D. But, unlike the NGF-effect, it was not associated with rapid induction of c-fos. © 1991.

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Molecular cloning of a cDNA of a camptothecin-resistant human DNA topoisomerase I and identification of mutation sites

Tamura H., Kohchi C., Yamada R., Ikeda T., Koiwai O., Patterson E., Keene J., Okada K., Kjeldsen E., Nishikawa K., Andoh T.

Nucleic Acids Research （Nucleic Acids Research）  19 （ 1 ） 65 - 75 1991年01月

研究論文（学術雑誌）, 共著, 査読有り,  ISSN  03051048

　概要を見る

Camptothecin (CPT), a plant alkaloid with antltumor activity, is a specific Inhibitor of eukaryotlc DNA topoisomerase I. We have previously isolated and characterized a CPT-resistant topoisomerase I isolated from a CPT-resistant human leukemia cell line, CPT-K5. cDNA clones of topoisomerase I were isolated from the CPT-resistant and the parental CPT-sensitive cell lines, respectively. Sequencing of the clones identified two mutations in the cDNA isolated from the resistant cells, which cause amino acid changes from aspartic acid to glycine at residues 533 and 583 of the parental topoisomerase I. When the CPT-K5 topoisomerase I was expressed in E. coli as a fusion protein with Staphylococcal Protein A fragment, the activity was resistant to CPT at a dose level up to 125 μM, whereas the parental fusion protein was sensitive to CPT as low as 1 μM. The resistance index (>125) of the CPT-K5 fusion topoisomerase I is similar to that of the native CPT-K5 topoisomerase I. These results indicate that either or both of the two amino acid changes identified in the mutant enzyme is responsible for the resistance to CPT. © 1991 Oxford University Press.

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Mode of Inhibition of Eukaryotic DNA Topoisomerase I Inhibitors

Tamura H., Ikegami Y.

nippon nogeikagaku kaishi （nippon nogeikagaku kaishi）  65 （ 6 ） 1008 - 1010 1991年

研究論文（学術雑誌）,  ISSN  00021407

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Acidic phospholipids directly inhibit DNA binding of mammalian DNA topoisomerase I

田村 悦臣

FEBS Letters （FEBS Letters）  261 （ 1 ） 151 - 154 1990年02月

研究論文（学術雑誌）, 共著, 査読有り,  ISSN  00145793

　概要を見る

Inhibition of mammalian DNA topoisomerase I by phospholipids was investigated using purified enzyme. Acidic phospholipids inhibited the DNA relaxation activity of topoisomerase I whereas neutral phospholipid, phosphatidylethanolamine, did not. Accumulation of a protein-DNA cleavable complex, an intermediate which is known to accumulate upon inhibition by a specific inhibitor camptothecin, did not occur. The filter binding assay revealed that the DNA binding activity of the enzyme was inhibited by acidic phospholipids. Moreoever, direct binding of phosphatidylglycerol to topoisomerase I was demonstrated. These results indicated that the inhibitory effect of acidic phospholipids on topoisomerase I was due to the loss of the DNA binding of the enzyme as a result of direct interaction between phospholipids and the enzyme. © 1990.

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Identification of the DNA binding subunit of RNA polymerase II from ehrlich ascites tumor cells

Horikoshi M., Tamura H., Sekimizu K., Obinata M., Natori S.

Journal of Biochemistry （Journal of Biochemistry）  94 （ 6 ） 1761 - 1767 1983年12月

研究論文（学術雑誌）, 共著, 査読有り,  ISSN  0021924X

　概要を見る

The DNA binding subunits of RNA polymerase II from Ehrlich ascites tumor cells were investigated in the following three ways.RNA polymerase II was dissociated in urea and the binding of the dissociated subunits to DNA was investigated.The RNA polymerase II: DNA complex was dissociated progressively with various concentrations of urea, and the subunits firmly attached to DNA were investigated.RNA polymerase II was dissociated into subunits in a SDS-polyacrylamide gel containing urea and blotted onto a nitrocellulose filter. The filter was then incubated with32P-nick-translated DNA to identify the DNA binding subunits.These procedures all showed that the largest subunit a of RNA polymerase II had strong affinity to DNA. It was found that a portion of subunits b and c could be recovered in DNA fraction when analyzed by procedures (1) and (2), but no significant DNA binding activity was detected when analyzed by procedure (3), suggesting that these subunits have either a much weaker affinity toward DNA compared to a or have affinity to a itself. © 1983 THE JOURNAL OF BIOCHEMISTRY.

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Differential expression of two abundant messenger RNAs during development of Sarcophaga peregrina

Tamura H., Tahara T., Kuroiwa A., Obinata M., Natori S.

Developmental Biology （Developmental Biology）  99 （ 1 ） 145 - 151 1983年09月

研究論文（学術雑誌）, 共著, 査読有り,  ISSN  00121606

　概要を見る

Expression of two abundant mRNAs in the fat body of third instar larvae of Sarcophaga peregrina was studied using genomic clones of storage protein and 25K protein as probes. It was found that these two genes were expressed sequentially in the third instar with a 20-hr interval between their expressions. No amplification of genes was detected during larval development, suggesting that the high level of mRNA was due to efficient transcription. The contents of the two mRNAs in third instar larvae were about the same, although the synthesis of storage protein was much more than that of 25K protein. © 1983.

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Tissue-Specific Distribution of DNA-Binding Nuclear Proteins

Tamura H., Natori S., Obinata M.

Differentiation （Differentiation）  23 （ 1 ） 87 - 90 1982年

研究論文（学術雑誌）, 共著, 査読有り,  ISSN  03014681

　概要を見る

The DNA-binding capacity of nuclear proteins of mouse cells was examined by the protein-blotting method. Under conditions in which the lac repressor specifically binds to the lac operator, the DNA-binding nuclear proteins from different tissues showed a tissue-specific distribution, suggesting that the species and amounts of nuclear proteins with DNA binding activity differ in different tissues. When cloned eukaryotic genes were used for binding, eukaryotic DNA showed stronger binding than prokaryotic DNA. Competition experiments suggested that many nuclear proteins have different DNA binding properties from that of the prokaryotic repressor. © 1982, International Society of Differentiation. All rights reserved.

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Purification of a new protein stimulating RNA polymerase II from ehrlich ascites tumor cells: Comparison with proteins purified before

Nakanishi Y., Sekimizu K., Tamura H., Natori S.

Journal of Biochemistry （Journal of Biochemistry）  90 （ 3 ） 805 - 814 1981年09月

研究論文（学術雑誌）, 共著, 査読有り,  ISSN  0021924X

　概要を見る

A protein factor named S-I(b) that stimulates the activity of RNA polymerase II was purified from Ehrlich ascites tumor cells. The final preparation gave a single band on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. This protein has a molecular weight of 24,000 and is significantly smaller than S-II and S-II', stimulatory proteins of RNA polymerase II purified previously. However, S-I(b) cross-reacted with antibody against S-II and its peptide map obtained after radio-iodination was identical with that of S-II' (a phosphorylated form of S-II), indicating that S-I(b) and S-II share a common primary structure. S-I(b) is suggested not to be a degradation product of S-II produced during the purification process, and the structural relations between S-II and S-II' are discussed. © 1981, by the Japanese Biochemical Society.

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Measurement of a stimulatory protein of RNA polymerase II in various mouse organs by the complement fixation test

Tamura H., Sekimizu K., Obinata M., Natori S.

Journal of Biochemistry （Journal of Biochemistry）  88 （ 5 ） 1475 - 1480 1980年11月

研究論文（学術雑誌）, 共著, 査読有り,  ISSN  0021924X

　概要を見る

The amount of S-II, a protein specifically stimulating RNA polymerase II, was measured in various mouse organs by a micro complement fixation assay. The amount was almost the same in brain, liver, kidney, spleen, and Ehrlich ascites tumor cells on the basis of DNA, being about 3.8 μg/mg DNA, which corresponds to 3.6 × 105molecules/cell. However, the amount of S-II decreased greatly during erythro-differentiation of Friend leukemia cells and no S-II was detected in matured erythrocytes. © 1980, by the Japanese Biochemical Society.

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