Ultrastructure and biochemical function of the mitochondria in respiratory-deficient mutant yeast induced by 4-nitroquinoline nitrogen oxide

1. A respiratory-deficient mutant strain of yeast was obtained from wild strain of Saccharomyces servisiae by treatment with 4-nitroquinoline N-oxide. Ultrastructure and function of the wild or mutant strains and the mitochondrial fractions isolated from these strains were examined by biochemical and electron microscopic analyses. 2. The frequency of the respiratory-deficient mutant strain in yeast induced with 10-6M 4-nitroquinoline N-oxide was about 40 %. 3. Respiratory-deficient mutant strain is incapable of reducing 2, 3, 5-triphenyltetrazolium chloride salt and to grow on lactate medium. In addition to this, the mutant has been found to have lost its ability to take up oxygen in sodium succinate and pyruvate. 4. 4.Nitroquinoline N-oxide in the concentration that induces a mutant of yeast cells or its kin inhibits the oxygen uptake in normal strain. 5. The normal strain of yeast is characterized by difference spectrum corresponding to cytochromes a+as, band c+Cll respectively, whereas, the mutant strain containes almost no cytochromes a+ as, band C1 but contains normal or increased amount of cytochrome c. 6. Mitochondrial fraction isolated from mutant strain has largely lost its ability to oxidize succinate. On the other hand, NADH-, lactate-and cytochrome c-oxidase activities are reduced by about 1/17, 1/7 and 1/8 of that of normal strain, respectively. 7. Succinate dehydrogenase activity of mutant strain is almost zero. Moreover, this activity is not affected on the addition of phenazine methosulfate. NADH dehydrogenase activity of mutant stran is about 1/2 of normal strain. 8. The variations in mitochondrial structure of normal and mutant strain in the stationary phase have been followed with the aid of electron microscopy. In contrast to the normal strain, the mutant strain revealed distinct morphological changes in mitochondria, especially, the lack of cristae in its interior. The results have been interpreted to indicate that the mutant induced by 4.nitroquinoline N.oxide has a character of cyto. plasmic mutant.</p

Activation and isolation of mitochondrial adenosine triphosphatase by ultrasonic irradiation

With the purpose to clarified the mode of localization and mechanisms of activation of ATPase in the mitochondrial membrane, analyses were made on the properties of mitochondrial ATPase from the structural and functional aspects. The activation of ATPase by DNP and Mg++ and the oligomycin sensitivity were investigated in a series of inner membrane fragment samples obtained by ultrasonic irradiation and those samples obtained in the processes of isolation and purification of ATPase from rat liver mitochondria and beef heart mitochondria in parallel with electron microscope observations. As a result it has been found that the membrane fragments obtained from rat liver and beef heart mitochondria by ultrasonication exhibited high respiratory activity and unmasked ATPase activity which was charac· terized by remarkable stimulation by Mg++ and inhibition by oligomycin and azide. Therefore, mitochondrial ATPase seems to be bound fairly closely to the inner mitochondrial membrane. In the membrane fragments prepared by ultrasonication of intact mitochondria, ATPase activity was stimulated by DNP, but in the supernatant fractions was not. On the other hand, the supernatant fraction obtained from BHM and inner membrane fragments by severe sonication exhibits a marked ATPase activity and the activity incresed in each step of the purification on the treatments with acid, protamine and heat. Especially in the case of membrane fragments the protamine treatment can be omitted. Electron microscope observation of the fractions in each step of the purification proved the head pieces to be ATPase. The ATPase activity of solubilized head pieces is insensitive to oligo. mycin and coincides with the soluble ATPase of PULLMAN etat. (8) in the points of its cold labile property and optimum pH, but it shown no accele. ration of ATPase activity by DNP.</p

Localization and Properties of DNA Molecules in the Mitochondria of Tumor Cells

1. Electron microscopic observations were made on DNA fibers in the mitochondria of tumor cells induced by various kinds of DNA viruses, RNA viruses, and chemical carcinogens, and also in those of normal and regenerating rat livers and cultured liver cells. Intramito-chondrial DNA fibers were observed most frequently in the sectioned specimens of adenovirus type 12-induced or SV 40-induced hamster tumor cells, but were hardly observed in normal and regenerating liver cells and other tumor cells. 2. The DNA fibers disappeared by treatment with DNase, but not with RNase. 3. The DNA fibers in adenovirus-induced tumor cells were easily isolated by osmotic shock and observed as circular DNA molecules by rotary shadowing with the electron microscope, while DNA molecules were hardly isolated from rat liver mitochondria by the same treatment. 4. On the ultracentrifugal fractionation of sonically disrupted adenovirus-induced tumor mitochondria, DNA were found to be contained mostly in the supernatant fraction by chemical analysis and by electron microscopic observation. 5. DNA fibers observed in the mitochondrial matrix in the sectioned specimens were proved to be identical with the isolated circular DNA molecules. 6. On the ultracentrifugal fractionation of sonically disrupted rat liver mitochondria, DNA were found to be contained mostly in the membrane fractions by chemical analysis. However, DNA molecules were difficult to observe with the electron microscope in the sectioned specimens of the rat liver mitochondrial membrane fractions. Nevertheless, they were isolated by phenol extraction from the membrane fractions, and were observed as circular DNA molecules with the electron microscope. 7. Appearance of the DNA fibers in the mitochondrial matrix seems to be associated with the division cycle of mitochondria, and in the mitochondria where DNA fibers are not observed in the sectioned specimens, the DNA molecules are supposed to be hidden by firmly attaching to the inner membrane, and to be isolated by chemical extraction

Ultrastructure and biochemical function of the mitochondria in respiratory-deficient mutant yeast induced by 4-nitroquinoline nitrogen oxide

1. A respiratory-deficient mutant strain of yeast was obtained from wild strain of Saccharomyces servisiae by treatment with 4-nitroquinoline N-oxide. Ultrastructure and function of the wild or mutant strains and the mitochondrial fractions isolated from these strains were examined by biochemical and electron microscopic analyses. 2. The frequency of the respiratory-deficient mutant strain in yeast induced with 10-6M 4-nitroquinoline N-oxide was about 40 %. 3. Respiratory-deficient mutant strain is incapable of reducing 2, 3, 5-triphenyltetrazolium chloride salt and to grow on lactate medium. In addition to this, the mutant has been found to have lost its ability to take up oxygen in sodium succinate and pyruvate. 4. 4.Nitroquinoline N-oxide in the concentration that induces a mutant of yeast cells or its kin inhibits the oxygen uptake in normal strain. 5. The normal strain of yeast is characterized by difference spectrum corresponding to cytochromes a+as, band c+Cll respectively, whereas, the mutant strain containes almost no cytochromes a+ as, band C1 but contains normal or increased amount of cytochrome c. 6. Mitochondrial fraction isolated from mutant strain has largely lost its ability to oxidize succinate. On the other hand, NADH-, lactate-and cytochrome c-oxidase activities are reduced by about 1/17, 1/7 and 1/8 of that of normal strain, respectively. 7. Succinate dehydrogenase activity of mutant strain is almost zero. Moreover, this activity is not affected on the addition of phenazine methosulfate. NADH dehydrogenase activity of mutant stran is about 1/2 of normal strain. 8. The variations in mitochondrial structure of normal and mutant strain in the stationary phase have been followed with the aid of electron microscopy. In contrast to the normal strain, the mutant strain revealed distinct morphological changes in mitochondria, especially, the lack of cristae in its interior. The results have been interpreted to indicate that the mutant induced by 4.nitroquinoline N.oxide has a character of cyto. plasmic mutant.</p

Imbalanced Hemolymph Lipid Levels Affect Feeding Motivation in the Two-Spotted Cricket, Gryllus bimaculatus.

Insect feeding behavior is regulated by many intrinsic factors, including hemolymph nutrient levels. Adipokinetic hormone (AKH) is a peptide factor that modulates hemolymph nutrient levels and regulates the nutritional state of insects by triggering the transfer of lipids into the hemolymph. We recently demonstrated that RNA interference (RNAi)-mediated knockdown of the AKH receptor (AKHR) reduces hemolymph lipid levels, causing an increase in the feeding frequency of the two-spotted cricket, Gryllus bimaculatus. This result indicated that reduced hemolymph lipid levels might motivate crickets to feed. In the present study, to elucidate whether hemolymph lipid levels contribute to insect feeding behavior, we attempted to manipulate hemolymph lipid levels via the lipophorin (Lp)-mediated lipid transferring system in G. bimaculatus. Of the constituent proteins in Lp, we focused on apolipophorin-III (GrybiApoLp-III) because of its possible role in facilitating lipid mobilization. First, we used RNAi to reduce the expression of GrybiApoLp-III. RNAi-mediated knockdown of GrybiApoLp-III had little effect on basal hemolymph lipid levels and the amount of food intake. In addition, hemolymph lipid levels remained static even after injecting AKH into GrybiApoLp-IIIRNAi crickets. These observations indicated that ApoLp-III does not maintain basal hemolymph lipid levels in crickets fed ad libitum, but is necessary for mobilizing lipid transfer into the hemolymph following AKH stimulation. Second, Lp (containing lipids) was injected into the hemolymph to induce a temporary increase in hemolymph lipid levels. Consequently, the initiation of feeding was delayed in a dose-dependent manner, indicating that increased hemolymph lipid levels reduced the motivation to feed. Taken together, these data validate the importance of basal hemolymph lipid levels in the control of energy homeostasis and for regulating feeding behavior in crickets