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.

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Ultra-thin serial sections of mitotic cells of Cryptococcus neoformans were observed by transmission electron microscope and computer-aided reconstruction of three-dimensional models were performed to understand the kinetics of the nucleus and mitochondria during mitosis. The separation of chromosomes occured in the protrusion of the nucleus extending in the bud (=daughter cell), followed by one set of chromosomes being moved back into the mother cell. These findings were similar to those reported in the heterobasidiomycetous yeasts. No giant mitochondrion, composed of a coalescence of all the mitochondria, was found. Volumetric analysis revealed the fluctuations in the mitochondrial volume: total cytoplasmic volume ratio were minimum during mitosis. In addition, methods, usefulness and limitations of observation of ultra-thin serial sections for understanding the morphology of cells and intracellular organelles were reviewed.

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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.

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Euglena cells were grown synchronously under photoautotrophic culture conditions on a 14h light-10h dark alternations. Changes in morphology of the pyrenoid and those in distribution of RuBisCO in chloroplasts were followed by immunoelectron microscopy during the growth and division phases of Euglena cells. The immunoreactive protein were densely localized in the pyrenoid, and thinly distributed in the stroma during the growth phase. During the division phase, the pyrenoid could not be detected and the gold particles were dispersed throughout the stroma. From a comparison of photosynthetic CO₂-fixation with the total carboxylase activity of RuBisCO extracted from Euglena cells in the growth phase, it is suggested that the carboxylase in the pyrenoid functions in CO₂-fixation in photosynthesis.
Cells of Euglena contain a LHC II. The precursors to LHC II are large polyproteins containing multiple copies of LHC II, and photocontrol of their formation is largely translational. Under conditions favoring LHC II accumulation in the thylakoids, a reaction with anti-LHC II antibody can be observed in the Golgi by immunogold electron microscopy. The timing of the immunoreaction in the Golgi in synchronous cells and in cells undergoing normal light-induced chloroplast development suggests that the nascent LHC II passes through the Golgi on the way to the thylakoids.

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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.

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There is no reference to the cell wall formation during the regeneration process in dermatophyte. Having succeeded in identifying the cell wall formation system of protoplast from Trichophyton mentagrophytes I introduce here the dynamics of the regeneration of this protoplast and the formation of cell wall using ultrahigh resolution low-voltage scanning electron microscopy and transmission electron microscopy (TEM), compare to the formation of yeast cell wall. Protoplasts were formed from mycelia of T. mentagrophytes; they regenerated the cell wall substances and were followed by a network of fibrils after 4-6hr, which developed into mature mycelia after 21hr. A network of twisted microfibrils and rope-shaped thick fiber was seen; it was covered with a sheath-shaped structure and was created on the surface of the developing mycelium. Intrafibrillar space and the surface of thick fibers were gradually filled with amorphous particles and a mature cell wall surface was formed. This protoplast regeneration was completely inhibited by the antifungal agent, lanoconazole (2ng/ml). The sectioned TEM image revealed thet normal mycelium having a thin cell wall developed from the abortive bead-shaped mycelium which was visible in the lose fibrous structure. Based on this result and our previos work on the ultrastructure of the mycelial surface of T. rubrum with lanoconazole, a model of the cell wall composition of Trichophyton is proposed.

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Preparations of human cervical mucus were obtained from fifteen female volunteers with or without vaginal candidiasis. Their content of lactoferrin and inhibitory effects on the growth of Candida albicans were estimated. The concentration of lactoferrin in cervical mucus was measured by enzyme linked immunosorbent assay (ELISA). Lactoferrin concentration of the eleven preparations among them was more than 0.2mg/ml. The effects of these cervical mucus preparations on Candida growth were examined in vitro. Candida growth was not inhibited by the addition of 1/200 diluted cervical mucus alone, however, anti-Candida activity of murine neutrophils was augmented by such an addition. These results suggest that the combination of neutrophils and cervical mucus may have an important role in defense against Candida infection in the vaginal mucosa.

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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.

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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 (C_MAX, 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.

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