The aim of this study was to determine the cellular consequences of the antifungal action of micafungin, voriconazole, amphotericin B, and liposomal amphotericin B. Therefore, we examined the morphological changes induced by fungicidal or fungistatic concentrations of these drugs on growing hyphae of Aspergillus fumigatus by using electron microscopy. Our results revealed that micafungin and voriconazole primarily affect cell wall formation, resulting in growth inhibition and cell lysis. In contrast, amphotericin B and liposomal amphotericin B affect the cytoplasmic membrane and the membranes of intracellular organelles, thereby leading lead to cell death.
True understanding of cell structure comes only from quantitative three-dimensional structural analysis. The term “structome”, coined by combining “structure” and “-ome”, is defined as quantitative three-dimensional structural information of whole cells at the electron microscopic level. We report here, for the first time, the structome of the cells of yeast Exophiala dermatitidis in the G1 phase determined using freeze-substitution and serial ultrathin sectioning electron microscopy. The cell wall and the nucleus occupied ～22% and ～7% of the cell volume respectively. There were 17-52 mitochondria per cell, occupying 7-12% of the cell volume. Five to ten endoplasmic reticula were present in a cell, occupying ～0.2% of the cell volume: they did not form a network in the cell. There were 1-4 vacuoles in a cell, occupying 4-10% of the cell volume. The Golgi apparatus, spindle pole body, autophagosomes, multivesicular bodies, lipid bodies, microtubules and microfilaments occupied ～1% of the cell volume in total. About 200,000 ribosome particles and 1,000 glycogen granules were present per cell. The cytosol occupied 43-53% of the cell volume. The membranes of cells of this yeast could be classified into three groups by their appearance and thickness.
Fourteen-membered-ring macrolides have been used widely against bacterial infection. These days, in addition to their well-established role as antibiotics, macrolides have an anti-inflammatory effect, in addition to their antibacterial effect, and are widely used at low dosages for long-term therapy for chronic inflammatory disease such as diffuse pan-bronchiolitis and chronic sinusitis. A macrolide-resistant coagulase-negative staphylococcal strain was obtained from the maxillary sinus of a patient with chronic sinusitis, who failed long-term macrolide therapy. Morphological observation demonstrated that this macrolide-resistant Staphylococcus capitis s strain had a thicker cell wall than macrolide-sensitive S. capitis strains. Moreover, macrolide-resistant four S. epidermidis strains isolated from patients also had thicker cell wall than macrolide-sensitive strain. On the other hand, the strain was not carrying any other than the four genes that are known mainly to encode for macrolide resistance in S. aureus, and it was not recognized in macrolide-resistant S. epidermidis strains that there was clear relationships between the genes encoding macrolide-resistance and the cell wall-thickness. Therefore, it was suggested that the strain had an unknown macrolide-resistance mechanism that might be related to cell wall thickening.
Staphylococcal γ-hemolysin (Hlg) consists of two separate proteins, Hlg1 (34kDa) and Hlg2 (32kDa), which lyses cooperatively mammalian erythrocytes. We have been studied pore-forming nature and assembly of Hlg on the membrane and revealed that Hlg1 and Hlg2 assemble alternately to form heteroheptameric transmembrane pores with subunit stoichiometries of 3:4 and 4:3. However, the three-dimentional (3-D) structure of Hlg pore complex has not been clarified yet. In this study, our aim is reconstruction of 3-D structure of the pore having a characteristic molecular arrangement in asymmetrical heteroheptamer, based on high-resolution electron transmission miscroscopic (TEM) images.
The shapes and sizes of subunit in the pore were measured on the basis of TEM image and the 3-D structure was constructed with computer-aided design software. As a result, it is estimated the pore forms a cylindrical structure in a superior region and funnel-shaped structure in an inferior region. That is, seven subunits bend to inside at the bottom of superior region and small cylindrical pore structure acting as functional transmembrane pore is attached. Additionally, it is revealed that several subunits are arranged out of order from vertexes of regular heptagon and largest mismatch subunit angle against the regular heptagon reachs approximately 15 degree.
The absorptive cells of the small intestine play an important role for nutrient digestion and absorption. In mammals, structural and functional changes occur twice at birth and weaning on the digestive tract including intestine. The absorptive cells vary with age and region of the intestine. During neonatal-suckling period, the absorptive cells have well-developed apical and basolateral endosomal network. The network is specialized for transcytosis in jejunum, for nutrient uptake and degradation in ileum and proximal large intestine. However, the apical endosomal network gradually disappears during weaning. After weaning, the absorptive cells only have basolateral endosomal network. Here, we review our data that the endosomal network of the intestinal absorptive cells and its transition from birth to weaning, and from proximal to distal.
Atomic force microscopy (AFM) has been used for imaging of nanoscale structures at solid/liquid interfarces due to its capability of operation in liquid. However, the performance of liquid-environment AFM has been significantly inferior to that of ultrahigh vacuum AFM that is used for atomic-scale imaging of surface structures and propoerties as well as for atom-by-atom manipulation. The atomic-scale imaging techniques used in ultrahigh vacuum has been realized by operating AFM in frequency modulation mode (FM-AFM) while the use of FM-AFM in liquid was considered to be very difficult. In 2005, however, a method to overcome this difficulty was developed and thereby true atomic resolution FM-AFM imaging in liquid was realized. This achievement triggered subsequent remarkable advances in liquid-environment AFM, enabling molecular- and atomi-scale imaging of biomolecules and hydration layers. In this article, basic principle of liquid-environment FM-AFM and its recent applications are described.
Cryo-electron microscopy (cryo-EM) is now being widely used to analyze biological macromolecular structures because it can be applied to various forms of samples, although attainable resolution is generally not so high. Helical reconstruction and single particle analysis of highly-symmetrical huge particles have now reached to a resolution better than 4Å, and allowed to visualize densities of amino-acid side chains and build the atomic models directly from the density maps. In this article, I will introduce examples of high-resolution cryo-EM structures such as the bacterial flagellar filament and a spherical virus, and review imaging conditions towards higher resolutions. The article also covers new digital imaging devices and energy filtering.
Owing to the advancement of electron microscope and the development of its related techniques, nanoscale structural information of amorphous structures has become obtainable by using spherical-aberration-corrected high resolution imaging and nanobeam electron diffraction. In addition, accurate radial distribution function analysis using energy filter has also become available to know averaged amorphous structures. In this lecture, we first explain the effectiveness of these techniques on the comprehensive structure analysis of metallic glasses, followed by introducing applications of these techniques especially to Pd and Fe-based metallic glasses.
Charge-coupled devices (CCDs) work on the principle of charge transfer. CCD image sensors have high sensitivity for the detection of almost single photons and low noise characteristics with excellent linearity between the input light power and the output signal. Therefore, they are widely used in microscopes, telescopes, and consumer video or still cameras. CCD image sensors output an image signal by using charges that are produced from the electron–hole pairs generated by light in the semiconductor, and the charges are transferred by the charge transfer method. The electron–hole pairs can also be generated by X-rays and electron beams. It is possible to obtain an electron image by direct electron irradiation of the CCD image sensor present inside a transmission electron microscope. In this lecture, the principle and structure of a CCD image sensor are described; its application when used inside a transmission electron microscope for obtaining images via direct single electron detection is also described.
Gastric proton pump, H+,K+-ATPase fulfills the remarkable task to generate very acidic environment (～pH 1) in the stomach lumen, which is indispensable for digestion and also act as first barrier against bacterial and viral infection. On the other hand, too much acid secretion induces acid-related diseases such as peptic ulcer. Therefore, the mechanism by which this massive H+ gradient generated is considerable biochemical interest. In order to address this mechanism, we have shown the three-dimensional structure of H+,K+-ATPase as determined by electron crystallography of two-dimensional crystals, using liquid-helium cooled cryo-electron microscopy. Here we introduce a molecular model for this remarkable biological phenomenon based on the EM structure of H+,K+-ATPase.
Dopnat clusters in silicon crystal has been reported to have an influence on the electronic properties. In order to clarify the existence of the cluster, it is necessary to observe it at a three-dimensional atomic level. Recently, aberration corrected electron microscope has been developed, which has been expected to improve not only transverse resolution but also depth resolution. Using our developed microscopy, R005, we found two dopant atoms facing each other in a six-member ring as the cluster.
Skyrmion is a novel more complex and nontrivial spin texture originally introduced as a model in nuclear physics to describe a localized particle-like configuration of field theory. Although the skyrmion in a magnetic solid is anticipated to produce unconventional spin-electronic functions such as the topological Hall effect, the skyrmion dynamics and its crystallography is not straightforward. To scrutinize the individual structure of skyrmion, we devised the skyrmion crystal (SkX) state as a two-dimensional (2D) magnet in a thin crystal of helical magnet Fe0.5Co0.5Si and performed the real-space observation by means of cryo-Lorentz transmission electron microscopy with a change of the magnetic-field lens current.