Inhibition of energy transfer reaction in mitochondria by the photosensitizing dye "NK19"--brief note

Among various photosensitizing dyes, 4, 4'-dimethyl 3, 3'-di-n-heptyl-8- {2-(4-methyl-3-n-heptylthiazole) }-2, 2'-dicarbocyanin diiodide (abb. NK19), even in an extremely low concentration, is known to inhibit the proliferation of bacteria and tissue culture cells (1, 2, 3). With respect to the mechanism of such inhibitory action no other property of this NKl9 is known except that it has a marked adsorptive property to protein (4). As a step toward the elucidation of the mode of biological effect, the author studied the effect of NK19 on the energy transfer reaction of Irat liver mitochondria, followed by comparison with the mode of actions of various other inhibitors of the oxidative phosphorylation (5). NK19. NKl9 can be prepared by letting 2, 4-dimethylthiazole heptyliodide react with ethylorthoformate in anhydrous acetic acid. We used NKI9, a product of Nihon Kanko Shikiso Research Laboratories. The molecular structure is as in the following and in its MeOH state it has maximum absorbancy at 590 m,a. For the use in experiment it was made into 1 mg/ml of MeOH and was stored in the dark until used.</p

Immunological study on cardiolipin from Escherichia coli

For the first time we found that cardiolipin was contained abundantly in Eschrichia coli, and we succeeded in isolating and purifying it as reported previously. With this E. coli cardiolipin a study was made on its reactivity to Wassermann antibody reagin by OGATA'S box titration, and the following results were obtained. L The purity of cardiolipin prepared from E. coli has been found to be satisfactory on the thin-layer chromatogram, by its chemical analyses and by its infrared spectrum study. 2. The composition of fatty acids of E. coli cardiolipin differed considerably from that of beef heart cardiolipin in the point that unsaturated fatty acids occupied only less than 66% in the former. Therefore, in the preparation of antigen, EtOH containing 20% tetrahydrofuran was used, which gave a clear solution, as E. coli cardiolipin did not dissolve completely in EtOH solution. 3. In the reaction made to take place with the serum from rabbit immunized with beef heart cardiolipin, E. coli cardiolipin gave almost the same reactivity to that of beef heart cardiolipin. 4. The reactivity of E. coli cardiolipin to the sera of syphilitic patients was also paretically the same as that of OGATA'S antigen, while it did not show any reactivity against the sera of normal person.</p

Lipid composition of adenovirus-induced tumor

The composition of total lipid extracted with chloroform-methanol from the AV12-induced tumor was investigated by thin-layer and paper chromatography. The content of lecitin and sphingomyelin was somewhat decreased and a cerebroside was characteristically detected in this tumor.</p

Freeze-etch studies on the bacterial cell surfaces: action of the cell wall lytic enzymes on the gram-positive cocci

Using a freeze-etching method, the ultrastructure of cell surface of gram-positive cocci was studied by digesting cell wall with lytic enzyme. In M. lysodeikticus, the cell surface revealed a very simplified ultrastructure, i. e. a single cell wall layer and a single plasma membrane layer. On the contrary, the cell surface of S. aureus exhibited a unique structure composed of two cell wall layers and a single ploasma membrane layer. The wall layers were constituted of 160 -180 A particle layer (CWl) which was unsusceptible to the L-ll enzyme and amorphous layer (CW2) which was susceptible. These results suggested that 160-180 A particles in CWl consisted mainly of the teichoic acid.</p

Selective staining of cytoplasmic membrane and nuclear apparatus of bacteria

A selective and simultaneous staining method for nuclear apparatus and cytoplasmic membrane of some bacteria has been presented. Nuclear apparatus is stained with basic fuchsin after hydrolysis with I N HCl and cytoplasmic membrane is restained with Victoria blue 4R after treating with saturated mercuric chloride. By this method, the nuclear apparatuses of B. subtilis, Sal. typhi 57 and Staph. aureus were stained red, and the cytoplasmic membrane and septum bluish purple distinctly. Thus this staining method would be of a great advantage in displaying the cellular structures of the bacteria.</p

The micromethod for determination of cholesterol, cholesteryl esters and phospholipids

We examined the method for determining microquantities of lipids, including cholesterol, cholesteryl esters and phospholipids. A standard colorimetric procedure of cholesteryl esters was modified to accommodate a quantitative thin-layer chromatography. This method involved the following steps. (1) Separation of lipids by a thin-layer chromatography: Lipids were applied to Silica gel G plates. Plates were developed with petroleum ether-diethyl etheracetic acid (82: 18: 2, vIvIv). (2) Elution of cholesterol and its esters from scraped silica gel: After scraping the silica gel with adhered cholesterol and its esters, they were eluted with chloroform-methanol (4: 1, v,tv). In the case of phspholipids, the silica gel was calcified. (3) Colorimetric determination of the lipids: Cholesterol and its esters eluted from the silica gel were determined by the method of ZAK with ROSENTHAL'S color reagent directly and after saponification, respectively. Phospholipids were calculated from the phosphorous content determined by the method of KATES. On the basis of examination of recovery and analyses of lipids extracted from tissue, it was concluded that this method permitted a reliable estimation of microquantities of cholesterol, its esters and phospholipids from small amounts of biological materials.</p

Alteration of phospholipid at various growth phases of Escherichia coli

By inoculating E. coli B into the semisynthetic medium we conducted shaking culture, and observed alterations of the total phospholipid contents and the amounts of individual phospholipid components in various stages of growth. The results are briefly summarized as follows. 1. The total phospholipid content has been found to be greater during early culture period, while it decreases as the growth age advances. 2. Phosphatidyl ethanolamine gradually increase as the culture period approaches the stationary phase. 3. Phosphatidic acid and phosphatidyl glycerol decrease precipitously as growth age advances. 4. Cardiolipin shows the maximum content in the middle log phase when the growth rate is most speedy.</p

A method of column chromatographic isolation of major phospholipid components on Escherichia coli

For the column chromatographic isolation of individual phospholipids from the total phospholipid mixture, silicic acid, DEAE cellulose, alumina and others, have been used as adsorbent. However, it must be emphasized that silicic acid (1, 2, 3, 4) is the most useful adsorbent for the separation of the total phospholipid mixture from each other in reasonable purity. VAN DEENEN reported that pure phosphatidyl glycerol was obtained from the lipid fraction of spinach leaves after repeated chromatography on silicic acid column (5). The phospholipid extracted from Escherichia coli B consists of abundant phosphatidyl ethanolamine (70-80 %), cardiolipin, phosphatidyl glycerol and other minor components as described in the previous paper (6). The high percentage content of phosphatidyl ethanolamine renders it difficult to separate the phospholipids by the column chromatography. Therefore, repeated chromatographies on the silicic acid column treated with sodium bicarbonate (7) and normal silicic acid column were employed for the isolation of the major components from the total phospholipid of E. coli B. Stepwise elution (4) was carried out with chloroform containing increasing proportions of methanol, and the eluent was divided into several fractions according to experience with thin-layer chromatography.</p

Ultrastructural alteration of the cell surface of Staphylococcus aureus cultured in a different salt condition

Staphylococcus aureus growing in a normal NaGI medium has a specific NaGI tolerance property to grow in the medium contain. ing NaGl in as high a concentration as over 10%. In our comparative study of the cells proliferating in the normal NaGI medium and 10% NaGl medium, we have observed the following differences aside from the changes of lipid composition in the cytoplasmic membrane previously reported. 1. S. aureus grown in high NaGl medium undergoes changes as to increase its size and reduce its surface area. 2. The thickness and weight of cell wall are increased to about 1. 7 times and 1. 32 times, respectively. 3. The protoplast prepared from S. aureus growing in the high NaGI medium shows a weaker resistance to hypotonic condition than that from normal cell.</p