Ultrastructural morphology is a principal study for almost all natural science, i. e., an inevitable and fundamental study to elucidate structures, functions and differentiation of a given tissue, cell, or molecule as a most understandable visual form. The number of young scientists, however, who have been engaged in this field is very small recently, since this field appears to be too old when compared with modern molecular biology and it takes much longer time for the beginner to master the methology for electron microscopy (EM). This symposium is designed for these young scientists and molecular biologists or biochemists, who are not so familiar to ultrastructural morphology, to better understand the applicability of EM, through new results and findings in ultrastructures of fungal cells and related organisms. EM includes several kinds of methods, which are shadowing EM, negative staining EM, ultrathin section EM, scanning EM, freeze-fracture EM, immuno-EM and diverse methods for staining the specimen. Shadowing EM and negative staining EM are suitable methods for the study molecular structures of proteins, and the former is prepared by shadowing with platinum palladium in a vacuum chamber, and the latter is a method to observe a relief prepared by dipping the sample in phosphotungsten solution. Freeze-fracture electron microscopy is suitable for the study of membrane plane ultrastructures, since it reveals a wide planar view of the membrane by splitting it along the hydrophobic membrane internal plane. Immunoelectron microscopy is an essential method for the study of intracellular localization of proteinous molecules. These methods will be introduced. This symposium will introduce new findings as for fungal cells, bacteria and protozoa obtained principally by using electron microscopy. These findings obtained through ultrastructures may provided a renewed knowledge of research approach from view points of ultrastructure.
This is a short review of fungal plasma membrane ultrastructure as revealed by freeze-replica. Characteristic features of growth polarity, growth phases, cell division, and ultrastructural characteristics after antibiotic treatments have been described.
The fine structure of the cell walls of Gram-positive and -negative bacteria were determined by electron microscopy with the new technique of freeze substitution method, and analysed the cell wall structure of Staphylococcus aureus in detail. The surface of Staphylococcal cell wall was covered with a fuzzy coat consisting of fine fibers or electron-dence mass. This coat was completely removed after extraction of teichoic acid from the cell wall with trichloroacetic acid treatment, but was not affected by sodium dodecyl sulfate or trypsin treatment. It was suggested that many amount of teichoic acid was located on the surface of the cell wall and less inside the cell wall. The capsule of strain Smith diffuse was assumed to play the role as the barrier protected from the penetration of antibody against teichoic acid.
We isolated two strains of Aspergillus flavus from a lung lesion and a skin lesion at autopsy from a patient with acute myelogenous leukemia complicated with fungal infection. An attempt was made to detect aflatoxins in culture filtrates of those isolates and the tissue extract of the lung lesion through the techniques of thin-layer chromatography (TLC), densitometry and high-performance liquid chromatography (HPLC). Aflatoxins B1, B2 and M1 were demonstrated in all of these materials qualitatively and quantitatively. The concentrations of aflatoxins in the cultures of the isolates and in the lung lesion extract determined by HPLC were aflatoxin B1: 11.715μg/ml (lung isolate), 21.383μg/ml (skin isolate), 0.635μg/g (lung extract), aflatoxin B2: 0.341μg/ml (lung isolate), 0.577μg/ml (skin isolate), 0.0273μg/g (lung extract) and aflatoxin M1: 0.277μg/ml (lung isolate), 0.491μg/ml (skin isolate), 0.0525μg/g (lung extract), respectively. B1, known as the most toxic among the aflatoxin group, showed the highest concentration through these experiments. This case may be considered as the first to detect aflatoxins in autopsied materials associated with A. flavus infection.
An automatic drug concentration simulator (DCS) has been developed and its applicability has been demonstrated by in vitro simulation of the human plasma concentration-time curve of fluconazole (FLCZ) against hyphal growth of Candida albicans and Aspergillus fumigatus. The response of hyphal growth to FLCZ was continually monitored and analyzed using an automatic hyphal growth analyzing system (Bio-Cell Tracer). The simulated concentration of FLCZ by DCS was confirmed by HPLC. The DCS assay was reproducible with a mean coefficient of variation (C. V., n=3) of 5.38%. When the growth of C. albicans hyphae was tested, there was a lag of onset of FLCZ effect between the time when FLCZ concentration became maximal (CMAX, 7.95μg/ml) and the point at which hyphal growth ceased. In contrast, FLCZ was found inactive against A. fumigatus. The newly devised technique could provide clinicians with important information in determining optimal dosing regimens for antifungal drugs.