Takuzo Oda

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.

Nemaline myopathy rod bodies

Ca2+-activated protease (CAF) digestion of glycerinated nemaline myopathy muscle removed the electron-dense material covering rods and Z-lines and exposed longitudinal backbone filaments, 6-7 nm wide, which span the lengths of the original rods. Decoration of the exposed filaments (which are responsible for the periodicity parallel to the long axis of intact nemaline rods) with heavy meromyosin (HMM) proved they are actin filaments. After CAF treatment, cross-striated periodical patterns in longitudinal sections and Z-filament-like proteins connecting actin filaments seen in cross-section disappeared. This suggests that alpha-actinin may be involved in formation of this pattern because of the specificity of CAF toward alpha-actinin. Gel electrophoresis of CAF-treated nemaline muscle showed that most alpha-actinin is released into the supernatant, whereas the residue is mainly actin and myosin. Electron microscope examination of longitudinal sections of intact rods shows an oblique filament pattern, thin (7 nm) lines, thick (11 nm) lines, and an amorphous-appearance previously observed in normal Z-lines, patterns observed depend on sectioning angle and section thickness. In cross-section, rods show small square net (SS) and basket-weave (BW) forms. The SS form predominates and coexistence of the 2 forms, which also occur in normal Z-lines, is observed. Results support the idea that rods are lateral polymers of Z-line units. We think that the length of rods, as well as the width of Z-lines, is determined by the amount of overlap of actin filaments of opposite polarity. Initiation of rod formation may be due to deregulation of actin filament length.

Determination of the concentration of adriamycin and its metabolites in the serum and tissues of Ehrlich carcinoma-bearing mice by high-performance liquid chromatography.

The concentration of adriamycin and its metabolites in serum and tissues was determined by high-performance liquid chromatography (HPLC) with a lapse of time after a single intraperitoneal administration to Ehrlich carcinoma-bearing mice. HPLC was carried out by using Zorbax Sil as the stationary phase and 3.8% sodium acetate in isopropanol as the mobile phase, at an excitation wavelength of 470 nm and an emission wavelength of 585 nm. Extraction of adriamycin using chloroform-methanol (4:1) gave recoveries of 98% from serum and 66-96% from tissues. Adriamycin rapidly disappeared from the serum, and the content of adriamycin per gram of tissue decreased in the order liver, duodenum, kidneys, spleen, lung, heart and tumour, and a high tissue retention occurred in the spleen and heart. The main metabolites of adriamycin were adriamycinone and adriamycinol, and other minor metabolites were detected.

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.

Determination of adriamycin (doxorubicin) and related fluorescent compounds in rat lymph and gall by high-performance liquid chromatography.

The concentrations of adriamycin (ADM) and related fluorescent compounds in lymph and gall were determined by high-performance liquid chromatography (HPLC) after a single intravenous injection into AH 109A tumour-bearing rats. HPLC was carried out by using Zorbax Sil as the stationary phase and chloroform--isopropanol--acetic acid--water--sodium acetate buffer (pH 4.5) (100:100:14:14:1) as the mobile phase, with a fluorescence spectrophotometric detector at an excitation wavelength of 470 nm and an emission wavelength of 585 nm. The detection limit for ADM was down to 1.0 ng/ml. In the thoracic duct lymph, the concentration of total ADM equivalent values (total ADM values) was maximal 30 min after injection and, after a subsequent decrease, increased gradually from 60 to 180 min. The ratio of total ADM in lymph to that in plasma at 180 min was 1.5 times that at 30 min. In gall, the total ADM showed a maximal level of 20.0 microgram/ml at 30 min.

Levels of topoisomerase II and DNA polymerase α are regulated independently in developing neuronal nuclei

A possible correlation between activity levels of topoisomerase II and DNA polymerase alpha was studied in neuronal nuclei from developing rat brain. A high level of canonical topoisomerase II activity was detected in neuronal nuclei throughout the development even at a late stage (28 days after birth) when the activity of DNA polymerase alpha decreased to less than 2% of the fetal level. Thus, in contrast to other systems, topoisomerase II does not change in parallel with DNA polymerase alpha during neuronal development. Our results suggest that topoisomerase II is required to maintain some fundamental processes in differentiated cells, including transcription.

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.

Studies on cytochrome oxidase: II. Ultrastructure of cytochrome oxidase☆

Abstract For the purpose of clarifying the ultrastructure of cytochrome oxidase, the fine structure of this entity was studied under various conditions. Fresh preparations of cytochrome oxidase, purified from beef heart mitochondria, consisted mainly of somewhat ellipsoid particles, measuring about 80–90 A in diameter. The particle assumed a cylindrical form measuring about 70 A in diameter at the base and 95 A in height under other conditions. Experimental results indicated that the particle was the smallest fundamental unit of active cytochrome oxidase. It was therefore designated as the unit particle of cytochrome oxidase (abbreviated as UPCO). The molecular weight of the particle, calculated from its volume and the average density (1.24) of lipoproteins (lipid:protein = 3:7), is about 270,000. The value is roughly twice the minimum molecular weight calculated from the heme a content. It is thus concluded that the unit particle contains two heme a and two copper atoms. The particles polymerized gradually during storage with a decrease in activity. The polymerization occurred most frequently in a linear fashion. In a sucrose gradient, ultracentrifugal analysis of a uniform preparation of cytochrome oxidase before and after treatment with SDS at an appropriate concentration, indicated that the particle located at the 22.6 S position was a dimer of the unit particle of cytochrome oxidase, while that located at the 5.7 S position was probably a half-unit particle. It appears also that the particle observed in the green membrane is a subunit (containing one heme a and one copper atom) of the unit particle of cytochrome oxidase and that the unit particle of cytochrome oxidase was constituted from two of the particles observed on the green membrane.

Two distinct cell lines derived from a human osteosarcoma

SummaryTwo cell lines were established from a human osteosarcoma transplanted into athymic nude mice after the second (O9N2) and fifth passages (HuO9). Both cell lines expressed 1,25(OH)2D3-responsive alkaline phosphatase activity and produced tumors in the dorsum of nude mice that were histologically similar to the original tumor. However, the morphological and growth characteristics of the two cell lines differed. O9N2 cells were large and polygonal, whereas HuO9 cells showed spindle shapes. HuO9 cells had a higher growth rate and saturation density than O9N2 cells. The c-myc oncogene was amplified 4-to 8-fold in HuO9 cells but not in O9N2 cells. Both cell lines had a homozygous internal deletion, lacking the 7.4-kb HindIII fragment in the Rb gene. The results suggest the importance of the c-myc oncogene in the growth and morphological control of human osteosarcoma cells and of the Rb gene in the pathogenesis of the tumor.