Akihiro Iino

Critical Point Drying Method Using Dry Ice

A critical point drying method using dry ice (solid carbon dioxide) instead of liquid carbon dioxide is reported. After the specimens are placed in the chamber of the medical point drying apparatus, dry ice cut to the shape of the chamber is inserted. The chamber is closed and warmed to change the dry ice into liquid carbon dioxide. This method needs no gas cylinder and even minute or fragile specimens do not blow away because there is no flow of gas. This method can be used with any kind of critical point drying apparatus.

Immunocytochemical localization of actin in the nucleolus of rat oocytes

Summary— In order to determine the localization of actin, growing and fully grown rat oocytes were immunocytochemically examined using a post‐embedding ultrastructural protein‐A gold technique. In quiescent oocytes, the nucleoplasm showed slightly lower levels of actin signal when compared to the surrounding cytoplasm. The highest levels of labeling were found on nucleoli showing a reticular type morphology. In oocytes at the diakinesis stage in which nucleolar compaction had occurred, the levels of labeling increased by 5–6 times those found in quiescent oocytes. Except for conspicuous accumulation of actin under the plasma membrane, compact nucleoli had significantly higher levels of labeling when compared with those found on the general cytoplasm, while the nucleoplasm with homogeneously dispersed chromatin showed significantly lower levels of associated actin signal than the general cytoplasm. In oocytes at metaphase I, the cytoplasmic region had comparable or lower levels of labeling than the cytoplasm of oocytes at diakinesis. The meiotic spindle embedded in material with medium electron density showed a similar level of labeling as the surrounding cytoplasm. On the other hand, significantly higher levels of associated actin were observed on the chromosomes of metaphase I. The actin signals were dispersed over the chromosomes and not concentrated on a specific region. These results suggest that nuclear actin may be involved in the process of chromosome construction and also the formation of the compacted structure of the nucleolus.

Impaired spermatogenesis and elevated spontaneous tumorigenesis in xeroderma pigmentosum group A gene (Xpa)-deficient mice

We have reported that xeroderma pigmentosum group A (Xpa) gene-knockout mice [Xpa (-/-) mice] are deficient in nucleotide excision repair (NER) and highly sensitive to UV-induced skin carcinogenesis. Although xeroderma pigmentosum group A patients show growth retardation, immature sexual development, and neurological abnormalities as well as a high incidence of UV-induced skin tumors, Xpa (-/-) mice were physiologically and behaviorally normal. In the present study, we kept Xpa (-/-) mice for 2 years under specific pathogen-free (SPF) conditions and found that the testis diminished in an age-dependent manner, and degenerating seminiferous tubules and no spermatozoa were detected in the 24-month-old Xpa (-/-) mice. In addition, a higher incidence of spontaneous tumorigenesis was observed in the 24-month-old Xpa (-/-) mice compared to Xpa (+/+) controls. Xpa (-/-) mice provide a useful model for investigating the aging and internal tumor formation in XPA patients.

Zur Frage der Verbindung der Linsenfasern im Rinderauge

Die Art der Verbindung zwischen den Linsenfasern im Rinderauge wurde im Auflicht- und Phasenkontrastmikroskop und anhand von Abdrucken mit dem Elektronenmikroskop untersucht.SummaryThe spatial distribution of the lens-fibers junction system was studied by means of incident light microscopy, phase contrast microscopy, and electron microscopy by replica method.The lens-fibers, especially in the cortex, are tightly connected with interdigitations on their sides and with many protoplasmic spines. These protoplasmic spines stretch themselves from each edge of the lens-fibers and penetrate into the clefts between the adjacent ones. Since such junction apparatus prevents the shifting of the lens-fibres, it is suggested that the morphological transformation of lens during accommodation is not provoked by lens-fibres displacement, but by the change of the content distribution in lens-fibers.Besides, it appeared that the shape of the lens-fibers is not regularly hexagonal but diversely deformed, with many protoplasmic spines.ZusammenfassungDie Art der Verbindung zwischen den Linsenfasern im Rinderauge wurde im Auflicht- und Phasenkontrastmikroskop und anhand von Abdrücken mit dem Elektronenmikroskop untersucht.Die Linsenfasern, besonders die des Cortex, sind miteinander verzahnt und durch Protoplasma-„Stacheln“ fest verbunden. Die Protoplasmastacheln stehen auf den sechs Kanten der im Querschnitt hexagonalen Linsenfasern und greifen in Fugen benachbarter Fasern ein. Diese Verbindung verhindert, daß sich die Linsenfasern gegeneinander verschieben. Die Formveränderung der Linse bei der Akkommodation muß also durch Änderungen in der Verteilung des Faserinhaltes hervorgerufen werden. Die Linsenfasern sind irregulär deformierte, stacheltragende Prismen von etwa hexagonalem Querschnitt.

The spatial distribution of spindle microtubules in anaphase 3Y1 cells.

Summary— The spatial distribution of the microtubules (MT) in the rat 3Y1 cells in mitosis was investigated by immunoelectron microscopy and computer‐graphic reconstruction of serial thin sections. In anaphase the interzone‐MT increased in number gradually with advancing phase, while the kinetochore‐MT in half‐spindles decreased. The interzone‐MT overlapped with each other at the equatorial region of the cell, and they formed a specific structure called the ‘stem bodies’. The ends of the interzone‐MT opposite to the stem bodies often attached to chromosomes but not to the poles. The stem bodies were not labeled with immunogold particles of anti‐α tubulin. Some of the stem bodies or MT which originate from stem bodies were found just beneath the plasma membrane in the equatorial region where abundant actin filaments appear showing the formation of the contractile ring and subsequently the cleavage furrow begins. On the basis of these observations it is assumed that the interzone‐MT is involved both in the separation of chromosomes in anaphase and in the formation of the cleavage furrow in telophase.

Diagnostic Revolution of Microhematuria by Real Time Confocal Scanning Laser Microscope: Hyodo-Iino-Miyagawa Method, Third Report

The real time confocal scanning laser microscope provides excellent three-dimensional images free of out-of-focus information. The objective of this study was to evaluate the usefulness of the laser microscope for the diagnosis of microhematuria. Characteristics of the test were evaluated in 81 patients with definite causes of hematuria. 30 erythrocytes in urinary sediments were examined for each patient, and those in whom less than 20% of the erythrocytes were poikilocytes were considered to have urological diseases and those in whom 80% or more of the erythrocytes were poikilocytes to have nephritis. According to these criteria, the sensitivity and the specificity of the examination to nephritis were 100 and 98.1%. 91.4% of the patients with urological disease had the nonglomerular type. The time required for the examination was less than 2-5 min in samples containing 1-3 erythrocytes in one field under an ordinary light microscope (x400).

Scanning and transmission electron-microscopic study of the development of the podocyte in the human fetus

Abstract. The metanephric kidneys of seven human fetuses at 11 – 17 weeks’ gestation were examined by scanning and transmission electron microscopy in order to evaluate differentiation of glomerular podocytes. When glomeruli were in the stage of S-shaped bodies, the surface of the visceral epithelium of renal corpuscles was smooth, with indistinct cell borders. As the glomeruli developed, visceral epithelial cells of renal corpuscles became spherical and resembled clusters of grapes in dense aggregation. In this stage, processes and foot processes were simultaneously formed at the base of epithelial cells. As glomeruli further differentiated, visceral epithelial cells of renal corpuscles began to separate from one another and became flat with the development of vascular loops. Processes and foot processes were exposed for the first time in Bowman’s space. In this stage, the degree of differentiation of epithelial cells varied widely among various sites of the glomerulus. As glomeruli developed further, projections became more complex and epithelial cells began to show structures similar to those of adult epithelial cells. The adjacent foot processes arose from different cells throughout the period of morphological differentiation.

Three-dimensional features of pancreatic cells

Almost 20 years have passed since the scanning electron microscope (SEM) was first applied to the biological field. In the beginning of the last decade, studies on the pancreatic cells with this instrument were limited to observing luminal and basal cell surfaces, since no feasible method to reveal internal cell structures existed. Observation of internal cell structures subsequently commenced with the development of various cracking methods [1–4]. However, progress in SEM in this field was delayed until quite recently due to difficulty in obtaining three-dimensional information on cell organelles prepared solely by the cracking method. Moreover, conventional SEM did not have enough resolving power to give distinct images of the fine details of cell organelles. As a result, there was little new discovery beyond the information already obtained through transmission electron microscopy (TEM).

A new concept of the pattern of structural changes with bone loss by histomorphometric analysis using bone slabs

Abstract: We examined naked bone slabs (1.2 mm thick) from iliac bone biopsied cores obtained from 33 women aged 33–89 years. The number, size, and shape of the pores in the bone slabs were analyzed. The results revealed that the % bone area (the percentage area occupied by bone in the slab) was linearly correlated with age and other parameters, such as the size of pores, irregularity of pores, and pore distance, but was not correlated to the number of pores. We found a second-degree polynomial relationship between the % bone area and the number of pores. Based on three parameters—% bone area, number of pores, and size of pores—cluster analysis was performed and the specimens divided into three groups. The group with sufficient bone mass showed few small round pores, and the group with severe bone loss revealed a few large pores that were caved in. The characteristics of these groups represented the relationship between bone mass and structural change. The remaining group with moderate bone loss was divided into two subgroups, one with an increased number of pores without expansion and one with expanded pores without an increase in number. We presumed that the variations between the groups were caused by differences between fine and rough structures in the trabeculae caused during the process of bone loss. We concluded that this analysis of bone slabs allowed the pattern of trabecular structural change that occurred with bone loss to be determined easily and visually.