Quantitative studies of nucleic acid in the cell by micro-spectrophotometry I. The critique and improvement of the microspectrophotometry

Some critical experiments have been carried out on the microspectrophotometry using the lymphocytes of a mouse, stained with Feulgen reaction, revealing that most reliable value can be attained by illuminating the material with a small spot-light and integrating the area surrounded by the extinction curve drawn by tracing along the diameter of the smeared and fixed cell.</p

Quantitative studies of nucleic acid in the cell by microspectrophotometry III. Nucleic acid contents in the cancer cells

1. The DNA contents in mature lymphocytes of the mouse, rat and man are kept almost constant. 2. The variety in the DNA contents in tumor cells is attributed to the rapid DNA synthesis taking place at the interphase, though the degenerating cells and the cells in abnormal mitosis can not be discarded as the source of the variety in DNA content. 3. The RNA content in AH-130 (ascites hepatoma) is less than that in normal liver cells.</p

Quantitative studies of nucleic acids in the cell with microspectrophotometer. II. Nucleic acid and hemoglobin contents in erythroid cells of frog and hen

Using the erythroid cells of Rana nigromaculata the hemoglobin synthesis has been studied in the relation of DNA and RNA contents. Results showed that the hemoglobin synthesis starts in the early stage of erythroblast but becomes marked just before the complete maturation. RNA contents drops markedly in the later stage of maturation. Measurement of DNA contents by Feulgen reaction suggested the termination of the mitosis just before the prematuration. From these results the author concludes that the RNA which will act as the template for the globin synthesis, develops from the early stage of erythroblast but the templation is accelerated in the terminal stage of maturation and the marked acceleration in hemoglobin synthesis in this stage.</p

Mitochondrial swelling induced by Ca2+ and inorganic phosphate and its related phenomena

Some investigations have been done on the relationships between the swelling-shrinkage change, oxygen consumption and state of oxidation-reduction of pyridine nucleotides of mitochondria, and between the swelling-shrinkage change of mitochondrial structure by Ca2+ and accumlation of Ca45 in rat liver mitochondria. A parallel relationship is observed between the Ca2+ induced swelling and Ca2+ accumulation. Both of them require Pi but not Mg2+, ATP and exogenous respiratory substrates and are inhibited by respiratory inhihitors or uncouplers of oxidative phosphorylation but not by the inhibitors of phosphorylating respiration. In this case the Ca2+ is transported with the phosphate even in ice cold. Even in the presence of antimycin A, moreover, Pi-dependent Ca2+ accumulation and Ca2+ induced swelling can be overcome by addition of ATP, which are inhibited by oligomycin. In the presence of Pi, mitochondria show shrinkage by addition of Ca2+ before the high amplitude swelling, which is closely correlated to the electron ransport chain and phosphorylation process of mitochondria, and the pattern of the mitochondrial shrinkage is quite similar to that observed in the case of respiratory control by ADP in intact mitochondria. This shrinkage of mitochondria is inhibited by respiratory inhibitor or uncoupler of oxidative phosphorylation but not by the inhibitor of phosphorylating respiration. From these data, therefore, it is considerd that the Ca2+ accumulation and Ca2+ induced shrinkage-swelling of mitochondria require the energy of oxidative phosphorylation with respect to the initial step before the oligomycin block.</p

Nucleoside-dependent synthesis of organic phosphorus compounds by rat liver nuclei

a) A modified procedure of the WIDNELL and TATA8 method yields rat liver nuclei manifesting a high degree of purity and activity. b) These nuclei contain a nucleoside-dependent phosphorylating activity that is readily released and apparently unrelated to either glycolysis or respiration. c) The main incorporation of the 32Pi is into ribose-I-phosphate; nucleoside phosphorylase activity satisfactorily accounts for the observed purine nucleoside stimulation of the nuclear phosphorus metabolism.</p

Growth inhibition and morphologic changes of HeLa cells exposed to unsaturated fatty acid fraction from the liver of x-ray irradiated rabbits (OX)

Biological effect of the unsaturated fatty acid fraction from the X-ray irradiated rabbit liver (OX) on HeLa cells has been observed in vitro comparing with the effect displayed on the same strain cells by the unsaturated fatty acid fraction from the non-irradiated rabbits, which is extracted by the same method as in OX, The observations have proven that OX is a powerful cytotoxin in a concentration of 0.05-0.025 per cent and induces a severe cell degeneration and cell death, resulting in a marked arrest in the growth of the cells. The similar effect has been observed by unsaturated fatty acid fraction from the nonirradiated rabbits, but the effect was much less comparing to that of OX. Possible mechanism of the cell damage by OX has been discussed.</p

An apparatus for simultaneous measurement of 90° light-scattering, fluorescence intensity of reduced pyridine nucleotides and oxygen consumption of mitochondria

1. An apparatus for the simultaneous measurements of volume change, fluorescence intensity of pyridine nucleotides and oxygen consumption of mitochondria has been constructed. 2. Oxygen consumption is measured by the rotating platinum electrode with a modification of Hagihara's system, attached in a cuvette of the apparatus. 3. Volume changes of mitochondria (swelling-shrinkage) are measured by the 90° light-scattering at 650 m&#956;. 4. Relative fluorescence intensity of pyridine nucleotides is measured by the fluorometer: for the excitation, a bright light at 365 m&#956;. line of mercury lamp is isolated through the filter and exposed to the mitochondria suspended in a cuvette of the apparatus, and fluorescent emission is analyzed by a grating mirror monochromator. 5. The scattered light at 650 m&#956;. is not affected by the excitation light and the fluorescent emission, and fluorescence intensity is not affected by the scattered light at 650 m&#956;. 6. The simultaneous measurements of the oxidation-reduction of pyridine nucleotides, the respiration states and the changes in the intensity of 90° lightscattering of mitochondria are given as an example of the performance of the present apparatus.</p

On the relation between the fatty acid composition and the swelling rate in rat liver mitochondria

1. For the purpose to clarify the relationship between the structural change and lipid composition of isolated rat liver mitochondria, lipid composition and swelling rate of mitochondria obtained from the rat of 3'-Me-DAB feeding and raised in cold room are measured, and the following results were obtained. 2. The mitochondria obtained from the liver of 3'-Me-DAB-fed rat and of rat raised in cold room show a low rate of swelling by addition of Na-oleate accompanied by the decrease in highly unsaturated fatty acids (C18:3 and C20:3or 4) and with the increase in saturated fatty acids (C16 and C18). 3. Activation energy for the mitochondrial swelling is about 16.2 Kcal in the mitochondria obtained from normal rat liver, but requires 19.7 Kcal in the mitochondria that show a low rate of swelling. The fatty acid composition, especially in glycerophosphatides which occupy about 80 per cent of total lipids, is a structural component of mitochondrial membrane, undergoes the change from former to latter in the following fashion: C16:0 21.73&#8594;32.10, C16:1 3.37&#8594;2.96, C18:0 25.0&#8594;29.75, C18:1 13.75&#8594;17.40, C18:2 23.90&#8594;16.0 and C20:3 or 4 12.23&#8594;1.79. 4. At the time of low rate swelling of mitochondria isolated from 3'-MeDAB- fed rat liver, there could be observed a marked increase of the acetone soluble lipid (simple lipids) in the total liver lipids and in the fatty acid distribution of the acetone-soluble lipids, oleic acid was markedly increased (0.838&#8594;3.81%/dry liver), despite the fact that in the acetone-insoluble fractions or in the mitochondria there are no marked changes in the oleic acid contents (1.84&#8594;2.56% or 0.212&#8594;0.246%/dry liver).</p

Relation between ANS fluorescence and energy states of mitochondria

1. ANS fluorescence change at various energized stages of mitochondria was investigated. 2. Freshly prepared mitochondria manifest ANS fluorescence change during anaerobic-aerobic transition, but aged and inner mitochondrial membrane show remarkable changes. 3. These data suggest that freshly prepared mitochondria or those in energized state exhibit less hydrophobic environments or decrease the binding site of ANS. 4. Energy dependent light scattering changes indicating the configurational changes of mitochondria cannot be said to be identical with the pattern of ANS fluorescence changes indicating the conformational change of mitochondrial membrane. 5. Polarity of the membrane structure and binding site of ANS in submitochondrial particles and mitochondrial membranes have been discussed.</p

Relation between mitochondrial swelling induced by inorganic phosphate and accumulation of P&sup3;&sup2; in mitochondrial Pi fraction

1. Rat liver mitochondria are swollen by inorganic phosphate in the medium of slightly hypotonic sucrose solution containing respiratory substrate and the mitochondrial swelling is inhibited or turned to shrink by ADP, respiratory inhibitor, anaerobiosis and uncoupler of oxidative phosphorylation. This mitochondrial swelling is not inhibited by the inhibitor of phosphorylating respiration such as oligomycin and tributyltin chloride. 2. Rat liver mitochondria are swollen by ATP in the presence of antimycin A, inorganic phosphate and 0.1 mM of CaCl2 and such a swelling is inhibited by oligomycin. 3. Accumulation of a small amont of P&#179;&#178; in acid soluble Pi fraction of rat liver mitochondria proceeds even in the medium containing neither ATP nor Ca++ but is inhibited by respiratory inhibitor, ATP, ADP and uncoupler of oxidative phosphorylation. The accumulation of P&#179;&#178; in mitochondria, however, is not inhibited by oligomycin. 4. The accumulation of P&#179;&#178; is induced by ATP in the presence of antimycin A and Ca++(O.l mM) and such an accumulation of P&#179;&#178; is inhibited by oligomycin. 5. It is suggested that the Pi-induced swelling of mitochondria is correlated to the accumulation of inorganic phosphate and both of them are tightly coupled to the initial step in the process of oxidative phosphoryaltion.</p