Electron microscopy of Pseudomonas aeruginosa treated with sulbenicillin and dibekacin.

A possible mechanism responsible for the combined effects of sulbenicillin and dibekacin on Pseudomonas aeruginosa IAM 1007 was investigated. The bactericidal activity of the above two drugs in combination was very strong. The regrowth of test strains after removal of the drugs was suppressed markedly, even when they were exposed to sulbenicillin plus dibekacin at a subinhibitory concentration of individual drugs. Sulbenicillin caused elongation of the bacterial cells. At the early stage of elongation, no demonstrable changes of ultrastructure of the cell wall were observed. At the late stage, lysis of the peptidoglycan layer occurred and spheroplast was formed. However, most of the outer membrane of the cell wall remained intact. Sulbenicillin acts upon the peptidoglycan layer, but not on the outer membrane. Thus it is difficult for sulbenicillin alone to cause cell lysis. On the other hand, dibekacin caused destruction of ribosomes and lysis of the outer membrane of the cell wall. Both sulbenicillin and dibekacin act on the cell wall, the former on the peptidoglycan layer (the inner membrane) and the latter on the outer membrane. The combined use of sulbenicillin and dibekacin caused elongation of bacilli and severe destruction of the inner and outer membranes of the cell wall. These morphological changes occurred even when the concentration of the individual drug was lower than its minimum inhibitory concentration (MIC). Furthermore, the cells elongated by sulbenicillin were ruptured easily when treated with dibekacin subsequently. The bacilli treated with dibekacin at a concentration lower than MIC and then treated with sulbenicillin at a concentration lower than MIC showed a marked elongation of the cells, which indicated that the effects of sulbenicillin was enhanced by dibekacin. These findings suggested strongly that sulbenicillin and dibekacin act on cell wall constituents and that their effects were complementary and synergistic.

Electron microscopic study of the cell surface of dibekacin-treated Pseudomonas aeruginosa.

Electron microscopy of thin sections of Pseudomonas aeruginosa IAM 1007 treated with dibekacin revealed blebs, disintegration of the outer membrane of the cell wall and degenerative features of the cytoplasm. In the next experiment, the cell wall fraction was isolated from the mechanically disrupted cells, incubated with dibekacin and was subjected to electron microscopy, in order to find a clue to the action mechanism of dibekacin on the cell wall of Pseudomonas aeruginosa IAM 1007. As a result, it was found that unidentified substances were released from the surface of the cell wall and that the outer membrane of the cell wall disappeared. The degree of changes of the cell wall ultrastructure was almost proportional to the length of incubation with dibekacin. These findings strongly suggest that dibekacin directly disintegrates the outer membrane of the cell wall of Pseudomonas aeruginosa IAM 1007.

Ultrastructure of the Lewis lung carcinoma.

ELectron microscopic observations were made on the Lewis lung carcinoma, which has frequently been used in various experiments. Although the tumor cells had desmosomes, interdigitation, microvilli and basement membranes which were of epithelial nature, they did not show either squamous or adenomatous differentiation. On the basis of microscopic and ultrastructural findings, this tumor falls into the category of large cell carcinomas. At the periphery of the tumor, sinusoidal clefts between the strands of tumor cells contained many red blood cells without fibrin. While perfect blood vessels were observed towards the center of the tumor, large necrotic areas were observed. Dilated large vessels possessed some endothelial fenestrations. Light endothelial cells were also observed, which were penetrated by red blood cells. Interstitial components were very scant and collagen fibres were frequently observed to be dissolved.

Electron microscopic observations on pulmonary connective tissue stained by Ruthenium Red.

Ultrastructural studies on human lung were performed with special attention to the interstitial acid mucopolysaccharides by Ruthenium Red staining and several enzyme digestion tests with Streptomyces hyaluronidase, chondroitinase ABC, chondroitinase AC, heparinase, trypsin and collagenase. Periodic lateral granules on the major cross bands of collagen fibrils and amorphous coats on them became visible by Ruthenium Red staining. The surface of elastic fibres, associated microfibrils, and some fine fibrils 10-20 nm in diameter were stained. Ruthenium Red also stained the surface of fibroblast and smooth muscle cells, basement membrane and filamentous long segments. In the interstructural space, granular substances 10-80 nm in diameter and fine filaments 3--4 nm thick, which formed a fine reticular network, were clearly observed. They were not visible on the usual thin section. The granular substances were located on the cross points of the fine filaments. They spread continuously and connected with each of the cells and extracellular structures in the pulmonary interstitium. The results of the enzyme digestion tests on the Ruthenium Red-positive material are discussed.

Cytoplasmic microtubules of rat ascites hepatoma cells.

Cytoplasmic microtubules of 4 strains of rat ascites hepatoma cells including YS, AH 66F, AH 130 and AH 100B were investigated electron microscopically. Microtubules were clearly demonstrated when the cells were fixed at 20 degrees C or 37 degrees C and stained by tannic acid. Morphology, localization and volume density (AH 130 greater than AH 66F greater than YS greater than AH 100B) of microtubules were examined comparatively in these 4 strains and correlation between microtubules and cell deformability was discussed.

[Cardiotoxic study of aclacinomycin A. Subacute cardiotoxic effect of aclacinomycin A in rats (author's transl)].

Subacut e cardiotoxic effect of aclacinomycin A or adriamycin given by daily intraperitoneal injections for 5 days was studied in rats by electrocardiography (ECG), blood biochemical analysis, light microscopy and electron microscopy. Dose levels of aclacinomycin A and adriamycin were 4 and 8 mg/kg, and 2 and 4 mg/kg, respectively. The two drugs caused severe body weight loss at a dose of 8 mg/kg and 4 mg/kg, respectively. Aclacinomycin A-treated rats at a dose of 4 mg/kg showed slight changes in ECG, whereas adriamycin-treated rats at the same dose showed a heart rate decrease, QRS duration and QT interval prolongation and R and S waves amplitude elevation. Blood biochemical changes caused by both drugs at a dose of 4 mg/kg were increased in lipoperoxide and alpha-hydroxybutyrate dehydrogenase activity. Aclacinomycin A gave slight ultrastructural changes in some cardiac cells such as formation of myelin figure and vacuolization in mitochondria. But adriamycin caused remarkable alterations such as degeneration and destruction of mitochondria, vacuolization of sarcoplasm and disappearance of myofilaments, which were often observed near the capillaries and nuclei. These results suggest that the two antibiotics caused cardiotoxicity by a similar mechanism. However, the damage produced by aclacinomycin A was milder than that of adriamycin.

Experimental pulmonary emphysema in Syrian hamsters.

Syrian hamsters were exposed to the aerosol of a 5% solution of papain in the presence of cysteine and ethylenediamine-tetraacetic acid. Pulmonary emphysema in hamsters was observed after exposure to papain for two to three hours. There was no change with time in the severity of emphysema from the second through the fourth weeks after exposure to papain. Pathogenesis of pulmonary emphysema was discussed in relation to proteolysis of glycosaminoglycan-protein complex.

Electron microscopy of C-particles and small vesicles in a transplantable rat ascites hepatoma-66F.

Transplantable rat ascites hepatoma AH-66F was examined by an electron microscope. C-particles were numerously observed in the intercellular spaces and on the cell surface. The size of these particles varied from 90 to 200 nm in diameter. Small vesicles were also observed. These vesicles ranged from 40 to 50 nm in diameter. Most of them were located near the group of C-particles. Occasionally, small vesicles were observed in the form of a rosette around the C-particles.

Aryl hydrocarbon hydroxylase in liver nuclei of C3H/He and DBA/2 mice.

Aryl hydrocarbon hydroxylase activity in liver nuclei from C3H/He mice is apparently increased by the administration of 3-methylcholanthrene, but the enzyme activity from the DBA/2 mice is not. On the other hand, by treatment of the mice with phenobarbital sodium, the increased activity in liver nuclei was observed in both strains of mice. There are approximately the same levels of the apparent Km for benzo[alpha]pyrene in liver nuclei from both strains of mice even after treatment with 3-methylcholanthrene, but different Km values for NADPH and NADH are observed between the constitutive and induced enzyme, showing 0.032 and 0.091 mM for NADPH, and 0.303 and 1.67 mM for NADH, respectively. Both 5, 6- and 7, 8-benzoflavones enhance the activity in the constitutive enzyme, but inhibit it in the induced enzyme non-competitively. Nicotinamide inhibits both enzyme activities in liver nuclei. Cyclohexene oxide and 1, 1, 1-trichloropropane oxide enhance the activity in the induced enzyme, but not in the constitutive enzyme in liver nuclei. The differences in the properties between the contitutive and induced enzymes and between the enzymes in microsomes and in nuclei from mouse liver were discussed briefly.