Shuji Seki

Studies on cytochrome oxidase: I. Fine structure of cytochrome oxidase-rich submitochondrial membrane☆

Abstract To elucidate the organization of cytochrome oxidase in the mitochondrial membrane, a study has been made on the correlation between the fine structure of a cytochrome oxidase-rich submitochondrial membrane (green membrane) and purified cytochrome oxidase. The green membrane was obtained directly from beef heart mitochondria or from the mitochondrial electron-transfer particles by treatment with deoxycholate and potassium chloride, followed by centrifugation. Heme a content of the green membrane isolated from beef heart mitochondria and from the electron-transfer particles were about 2 nmoles per milligram protein and 3–4 nmoles per milligram protein, respectively. Electron micrographs of the green membrane revealed regular arrays of small particles on the surface of the membrane, each particle measuring approximately 50–60 A in diameter with a center-to-center distance of about 70 A. These particles were extracted from the green membranes and recovered in a purified cytochrome oxidase fraction. Membrane structure was re-formed with the purified cytochrome oxidase, and the surface of the reconstituted membrane exhibited a particulate structure similar to the original green membrane. These results indicate that there is a correlation between the particles on the green membrane and cytochrome oxidase.

A mouse DNA repair enzyme (APEX nuclease) having exonuclease and apurinic/apyrimidinic endonuclease activities : purification and characterization

A mouse repair enzyme having priming activity on bleomycin-damaged DNA for DNA polymerase was purified to apparent homogeneity and characterized. The enzyme extracted from permeabilized mouse ascites sarcoma (SR-C3H/He) cells with 0.2 M potassium phosphate buffer (pH 7.5) was purified by successive chromatographies on phosphocellulose, DEAE-cellulose, phosphocellulose (a second time), Sephadex G-100, single-stranded DNA cellulose and hydroxyapatite. The purified enzyme has an Mr of 34,000 as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Enzymatical studies indicated that it is a multifunctional enzyme having exonuclease, apurinic/apyrimidinic endonuclease and phosphatase activities, similar to Escherichia coli exonuclease III. This enzyme is tentatively designated as APEX nuclease for apurinic/apyrimidinic endonuclease and exonuclease activities. The amino acid composition, amino-terminal amino acid sequence and an internal amino acid sequence of APEX nuclease are determined.

Differential effects of aphidicolin on replicative DNA synthesis and unscheduled DNA synthesis in permeable mouse sarcoma cells

Aphidicolin clearly discriminated replicative DNA synthesis from unscheduled DNA synthesis. Aphidicolin inhibited replicative DNA synthesis in permeable mouse ascites sarcoma cells. The mode of inhibition of aphidicolin was a mixed type with respect to deoxycytidine triphosphate but was non-competitive with respect to the other three deoxynucleoside triphosphates. Aphidicolin did not affect the activity of unscheduled DNA synthesis in either bleomycin-treated permeable sarcoma cells or isolated rat liver nuclei. Considering the difference in sensitivity of DNA polymerase alpha and beta to aphidicolin, and other related information reported previously, the results are compatible with the idea that DNA polymerase alpha is involved in replicative DNA synthesis and DNA polymerase beta in unscheduled DNA synthesis in the present systems.

Disruptive forces for swine heart, liver, and spleen : Their breaking stresses

OBJECTIVES The mechanical strengths of the swine heart, liver, and spleen were determined by measuring the force that initiated the rupture of the organs. METHODS The force was loaded by the compression of a detection bar onto the organ, and the breaking stress was calculated by dividing the force by the bars contact area. The forces were measured with five measurements in five of each type of organ with and without the serous membrane (SM). RESULTS The breaking stresses of the heart were 14.1+/-0.7 kg/cm2 (mean+/-SEM) with intact epicardium and 8.1+/-0.6 without it; those of the spleen were 9.0+/-0.9 with its capsule and 1.8+/-0.1 without the capsule, and those of the liver were 4.6+/-0.3 with and 3.6+/-0.3 without the SM. A three-way layout analysis of variance showed that the organs, their SM and individual swine were independent determinants of the breaking stresses. CONCLUSION With the intact SM, the heart was strongest and the liver was weakest. When the SM was not present, the spleen lost 80% of its strength, the liver 22% of its strength, and the heart lost 43% of its strength.

Accumulation of wild-type p53 in astrocytomas is associated with increased p21 expression

Abstract Approximately one quarter of human astrocytomas show immunohistochemical positivity for p53 protein but lack p53 gene mutations, which could reflect either an accumulation of wild-type p53 protein or an inadequate sensitivity of mutation detection. Since wild-type p53 up-regulates p21 expression, increased p21 expression in those astrocytomas with p53 accumulation in the absence of mutations would argue that the protein was wild type in these tumors. We therefore compared p21 expression with p53 gene and protein status in 48 primary human astrocytomas. Single-strand conformation polymorphism analysis and direct sequencing of the p53 gene showed mutations in 11 tumors (22.9%), while immunohistochemistry revealed positive staining in 19 cases (39.6%). Those tumors with p53 immunopositivity in the absence of p53 mutation had significantly increased p21 expression when compared to either mutant p53 or p53-immunonegative cases. Neither p53 nor p21 status correlated with proliferation indices, as assessed by Ki-67 immunohistochemistry. These results support the hypotheses that functionally wild-type p53 accumulates in some astrocytomas, and that alternative cell cycle checkpoints (such as the p16 pathway) may be more important than p21 in regulating proliferation in astrocytomas.

Relationship between expression of a major apurinic/apyrimidinic endonuclease (APEX nuclease) and susceptibility to genotoxic agents in human glioma cell lines.

The multifunctional DNA repair enzyme (APEX nuclease) having apurinic/apyrimidinic (AP) endonuclease, 3′-5′ exonuclease, DNA 3′ repair diesterase and DNA 3′-phosphatase activities is thought to be involved in repair of AP sites and single-strand breaks with 3′-blocked termini. To investigate the biological role of the enzyme, we studied the correlation between APEX AP endonuclease activity in several human glioma cell lines having various degree of its expression and cellular susceptibility to cytotoxic agents such as methyl methanesulfonate (MMS), 1-(4-amino-2-methyl-5-pyrimidinyl)methyl-3-(2-chloroethyl)-3-nitrosourea hy-drochloride (ACNU), cis-diamminedichloroplatinum(II) (CDDP), etoposide (VP-16), hydrogen peroxide (H2O2), hyperthermia and X-ray. The cell lines having lower APEX expression showed higher sensitivity to MMS and H2O2 which are known to induce AP sites and single strand breaks on DNA, respectively. The cellular susceptibility to the other agents tested was not significantly correlated to the APEX expression. The present results are thought to support the notion that APEX nuclease plays an important role on repair of AP sites and single-strand DNA breaks with 3′-blocked termini in mammalian cells.

Identification of the functional elements in the bidirectional promoter of the mouse O-sialoglycoprotein endopeptidase and APEX nuclease genes.

The gene for mammalian O-sialoglycoprotein endopeptidase (Osgep) lies immediately adjacent to the gene for the APEX nuclease (Apex), a multifunctional DNA repair enzyme, in a head-to-head orientation. To clarify the regulation of these divergent genes, we studied their promoter regions with luciferase reporters. Deletion analysis of a fragment containing the entire mouse Apex gene suggested that cis-acting elements driving in the direction of Osgep are widely distributed in the mApex gene, in the antisense orientation. We investigated in detail cis-acting elements near the transcription initiation site of mOsgep. The spacer sequence between mOsgep and mApex was shown to have bidirectional promoter activity and it has been reported that two CCAAT boxes promote basal transcription in the direction of mApex. However, only one of the CCAAT boxes proximal to the transcription initiation site of mOsgep was important for transcription towards mOsgep. An Sp1-binding sequence was found to be involved in bidirectional transcription and a CRE/ATF-like sequence was shown to function as a repressor of mOsgep transcription. Quantitative RT-PCR showed that the mApex and mOsgep genes were expressed in all tissues examined and that expression of mOsgep was low compared with mApex.

DNA synthesis in detergent-treated mouse ascites sarcoma cells

Mouse ascites sarcoma cells (SR-C3H/He cells) were made permeable to nucleoside triphosphates by treatment with nonionic detergents in a nearly isotonic condition. The permeable cells synthesized DNA in the presence of the four deoxyribonucleoside triphosphates, ATP, Mg2+, and the proper ionic environment. The optimum detergent concentration for DNA synthesis was 0.015--0.020% with Triton X-100, 0.020% with Nonidet P-40, and about 0.0025% with Brij 58. Higher concentrations of detergents were rather inhibitory to DNA synthesis. DNA synthesis in Triton-permeabilized cells was thought to be replicative, and the activity in the optimum conditions was much higher than that measured in hypotonic permeable cells or in isolated nuclei. These studies show the potential usefulness of detergent treatment for examining DNA replication in mammalian cells in vitro.

Effect of aurintricarboxylic acid on in vitro DNA synthetic activity of mouse ascites sarcoma cells

Abstract Aurintricarboxylic acid inhibited replicative DNA synthesis in nucleotide-permeable mouse ascites sarcoma cells. DNA polymerase activity assayed with activated DNA template and DNA polymerase purified partially from sarcoma cells was also inhibited by aurintricarboxylic acid. The inhibition of DNA polymerase activity was probably due to the inhibitory interaction of aurintricarboxylic acid with DNA polymerase. The replicative DNA synthesis might be inhibited by aurintricarboxylic acid interacting with some essential protein component(s), such as DNA polymerase of the replication machinery.

Detection of possible DNA repair enzymes on sodium dodecyl sulfate-polyacrylamide gels by protein blotting to damaged DNA-fixed membranes

A novel method for detecting possible DNA repair enzymes on sodium dodecyl sulfate-polyacrylamide gels by blotting them onto a damaged DNA-fixed membrane is presented. To prepare the membrane, highly polymerized calf thymus DNA immobilized on a nylon membrane is damaged chemically. Enzymes, either homogeneous or crude, that are possibly involved in the priming step of DNA repair are fractionated by SDS-polyacrylamide gel electrophoresis (SDS-PAGE) and are renatured to active form by incubating the gel in an appropriate buffer. The renatured enzyme is then blotted onto the damaged DNA-fixed membrane, a process during which incision and/or excision are introduced to the damaged DNA by the enzymes. The incision and/or excision provide priming sites for repair DNA synthesis in the subsequent step in which the membrane is incubated with DNA polymerase in the presence of alpha-32P-labeled substrate. The site of substrate incorporation on the membrane reflecting the molecular weight of the repair enzyme is finally visualized by autoradiography. The present technique is established using Escherichia coli exonuclease III and a DNA-fixed membrane treated with bleomycin or acid-depurinated. By application of this method, a priming factor (an exonuclease) involved in the initiation of bleomycin-induced DNA repair is detected in the extract of mouse ascites sarcoma cells, and thus the molecular weight of the enzyme is estimated. Some apurinic/apyrimidinic endonucleases of mammals are also detected by the present procedure.