Insulin balls, localized insulin amyloids formed at the site of repeated insulin injections in patients with diabetes, cause poor glycemic control and cytotoxicity. Our previous study has shown that ...insulin forms two types of amyloids; toxic amyloid formed from the intact insulin ((i)-amyloid) and less-toxic amyloid formed in the presence of the reducing reagent TCEP ((r)-amyloid), suggesting insulin amyloid polymorphism. However, the differences in the formation mechanism and cytotoxicity expression are still unclear. Herein, we demonstrate that the liquid droplets, which are stabilized by electrostatic interactions, appear only in the process of toxic (i)-amyloid formation, but not in the less-toxic (r)-amyloid formation process. The effect of various additives such as arginine, 1,6-hexanediol, and salts on amyloid formation was also examined to investigate interactions that are important for amyloid formation. Our results indicate that the maturation processes of these two amyloids were significantly different, whereas the nucleation by hydrophobic interactions was similar. These results also suggest the difference in the formation mechanism of two different insulin amyloids is attributed to the difference in the intermolecular interactions and could be correlated with the cytotoxicity.
Several types of macrophages have been reported in the intestinal mucosa, but their histological localization remains ambiguous. Here, we obtained detailed information about ultrastructural and ...phenotypical diversity of macrophage-like cells (MLCs) in the rat ileal mucosa using immunofluorescent analysis and serial block-face scanning electron microscopy (SBF-SEM). The results revealed that the cells immunopositive for CD68, the pan-macrophage marker, included CD163
−
CD4
+
, CD163
+
CD4
+
, and CD163
−
CD4
−
cells in the lamina propria (LP) of the intestinal villus and around the crypt. CD68
+
CD4
+
CD163
−
cells seemed to be preferentially localized in the intestinal villus, whereas CD68
+
CD163
+
CD4
+
cells were frequently localized around the crypt. SBF-SEM analysis identified three types of MLCs in the ileal mucosa, which were tentatively named types I–III MLC based on aspects of the 3D-ultrastructure, such as the localization, quantity of lysosomes, endoplasmic reticulum, and exoplasm. Type I and II MLCs were localized in the villous LP, while type III MLCs were localized around the crypt, although type II MLCs were a minor population. All three MLC types extended their cellular processes into the epithelium, with type I MLCs showing the greatest abundance of extended processes. Type I MLCs in the upper portion of the intestinal villus showed a higher level of attachment to intraepithelial lymphocytes (IELs) compared to type III MLCs around the crypt. These findings suggest that macrophages of the rat ileal mucosa differed by region along the longitudinal axis of the villous tip-crypt from the perspective of ultrastructure, cellular composition, localization, and interactions with IELs.
Eosinophils are abundantly present in intestinal mucosa. However, the morphological characteristics of their cellular population are still largely unknown. In this study, we examine their ...characteristics in the rat ileal mucosa using histological and ultrastructural methods. The results indicated that ileal eosinophils could be distinguished into two main groups based on their nuclear shapes and distribution: eosinophils with spheric or reniform nuclei mainly localized in the villous region and eosinophils with annular or bacilliform nuclei as the major population around crypts. Immunohistochemical analysis revealed that all eosinophils in the lamina propria (LP) were immunopositive for CD11b, whereas eosinophils in LP of the intestinal villus but not those in LP around the crypt, were immunopositive for CD11c. Three-dimensional ultrastructural analysis using serial block-face scanning electron microscopy showed that the eosinophils with spheric or reniform nuclei were abundant in the upper portions of the intestinal villus, whereas those with annular nuclei were abundant in the lower portions of the intestinal villus and around crypts. The eosinophils with spheric or reniform nuclei possessed broader cellular bodies with greater abundance of surface projections compared with those with annular nuclei. Eosinophils in the upper portions of intestinal villus frequently extended their cellular bodies into the intraepithelial space. The number of total and eosinophil-specific granules was positively correlated with the minor axis of the nuclear holes in the annular nuclei. These data suggest that ileal eosinophils exhibit not homogenous but rather diverse characteristics, possible due to the mixture of eosinophils at different maturation and/or activation stages.
The comprehensive targets of innervation in the intestinal mucosa are unknown, partly because of the diversity of cell types and the complexity of the neural circuits. Herein, we investigated the ...comprehensive targets of neural connectivity and analyzed the precise characteristics of their contact structures in the mucosa of rat ileum. We examined target cells of neural connections and the characteristics of their contact structures by serial block-face scanning electron microscopy at four portions of the rat ileal mucosa: the apical and basal portions in the villi, and the lateral and basal portions around/in the crypts. Nerve fibers were in contact with several types of fibroblast-like cells (FBLCs), macrophage-like cells, eosinophils, lymphocyte-like cells, and other types of cells. The nerve fibers almost always ran more inside of lamina propria than subepithelial FBLC, and thus contacts with epithelial cells were very scarce. The contact structures of the nerve fibers were usually contained synaptic vesicle-like structures, and we classified them into patterns based on the number of nerve fiber contacting the target cells at one site, the maximum diameter of the contact structures, and the relationship between nerve fibers and nerve bundles. The contact structures for each type of cells occasionally dug into the cellular bodies of the target cells. We revealed the comprehensive targets of neural connectivity based on the characteristics of contact structures, and identified FBLCs, immunocompetent cells, and eosinophils as the candidate targets for innervation in the rat ileal mucosa.
The present study aimed to histologically clarify the regional specificity of the mucosal enteric glial cells (mEGCs) in the rat intestine with serial block-face scanning electron microscopy ...(SBF-SEM). SBF-SEM analysis with the ileum, the cecum and the descending colon revealed that mEGC nuclei were more abundant in the data stacks from the apical portion of the villus and the lateral portion of the crypt of the ileum. mEGCs exhibited a high rate of coverage over the nerve bundle around the lateral portion of the ileal crypt, but showed an extremely low level of coverage in the luminal portion of the cecum. These findings evidenced regional differences in the localization of mEGCs and in their sheath structure in the rat intestine.
Serial block-face scanning electron microscopy (SBF-SEM) is useful for three-dimensional observation of tissues or cells at high-resolution. In this study, SBF-SEM was used to three-dimensionally ...analyze the characteristics of fibroblast-like cells (FBLCs) in the rat ileal lamina propria (LP). The results revealed that FBLCs in LP could be divided into four types, tentatively named FBLC type I-IV, based on the external cellular appearance, abundance or shape of each organelle, detailed distribution in the LP and relationship with surrounding cells. FBLC-I and -II localized around the intestinal crypt (InC), FBLC-III localized from the lateral portion of InC to the apical portion of the intestinal villus (InV), and FBLC-IV localized in the InV. FBLC-I, -II and -III, but not FBLC-IV, localized beneath the epithelium. FBLC-II possessed thin lamellar-shaped endoplasmic reticula, whereas FBLC-I possessed expanded endoplasmic reticula that occasionally showed a spherical shape. FBLC-III and -IV possessed a cytoplasmic region with high-electron density and no organelles immediately beneath the cellular membrane; this region was found at the epithelial sides in FBLC-III and scattered in FBLC-IV. FBLC-IV were in constant close proximity to villous myocytes throughout the InV and also in contact with FBLC-III especially in the apical portion of the InV. FBLC-I, -II and -IV, but not -III, were constantly found to be in contact with various immunocompetent cells in the LP. Three-dimensional analysis using SBF-SEM indicates that four types of FBLC localized in the rat ileal LP.
Paramyosin is a myosin-binding protein characteristic of invertebrate animals, while troponin is a Ca
-dependent regulator of muscle contraction. Both proteins are widely distributed in protostomes, ...while in deuterostomes, their distribution is limited; namely, presence of paramyosin and absence of troponin are common features in echinoderm muscles, while muscles of chordates contain troponin but lack paramyosin. In this study, we examined the muscle of a hemichordate, acorn worm, to clarify whether this animal is like echinoderms or like the other deuterostome animals. We found a 100-kDa protein in the smooth muscle of acorn worm. This protein was identified with paramyosin, since the purified protein formed paracrystals with a constant axial periodicity in the presence of divalent cations as paramyosin of other animals, showed ability to interact with myosin and shared common antigenicity with echinoderm paramyosin. On the other hand, troponin band was not detected in isolated thin filaments, and the filaments increased myosin-ATPase activity in a Ca
-independent manner. The results indicate that troponin is lacking in thin filaments of acorn worm muscle just as in those of echinoderms. The muscle of hemichordate acorn worm is quite similar to echinoderm muscles, but different from chordate muscles.
In this study, we investigated use of local surface plasmon resonance (LSPR) of metal nanoparticles (NPs) as a correlative light and electron microscopy (CLEM) tag for biological samples. Gold NPs in ...ultra-thin sections for TEM revealed that LSPR could be observed by optical microscopy at sizes of 20 nm or larger. Gold NPs at sizes less than 20 nm could be observed using the gold enhancement method. Therefore, this CLEM tag could be applied to immunoelectron microscopy using this gold enhancement method.
A hippocampal mossy fiber synapse, which is implicated in learning and memory, has a complex structure in which mossy fiber boutons attach to the dendritic shaft by puncta adherentia junctions (PAJs) ...and wrap around a multiply‐branched spine, forming synaptic junctions. Here, we electron microscopically analyzed the ultrastructure of this synapse in afadin‐deficient mice. Transmission electron microscopy analysis revealed that typical PAJs with prominent symmetrical plasma membrane darkening undercoated with the thick filamentous cytoskeleton were observed in the control synapse, whereas in the afadin‐deficient synapse, atypical PAJs with the symmetrical plasma membrane darkening, which was much less in thickness and darkness than those of the control typical PAJs, were observed. Immunoelectron microscopy analysis revealed that nectin‐1, nectin‐3, and N‐cadherin were localized at the control typical PAJs, whereas nectin‐1 and nectin‐3 were localized at the afadin‐deficient atypical PAJs to extents lower than those in the control synapse and N‐cadherin was localized at their nonjunctional flanking regions. These results indicate that the atypical PAJs are formed by nectin‐1 and nectin‐3 independently of afadin and N‐cadherin and that the typical PAJs are formed by afadin and N‐cadherin cooperatively with nectin‐1 and nectin‐3. Serial block face‐scanning electron microscopy analysis revealed that the complexity of postsynaptic spines and mossy fiber boutons, the number of spine heads, the area of postsynaptic densities, and the density of synaptic vesicles docked to active zones were decreased in the afadin‐deficient synapse. These results indicate that afadin plays multiple roles in the complex ultrastructural morphogenesis of hippocampal mossy fiber synapses.
A hippocampal mossy fiber synapse has a complex structure, in which mossy fiber boutons attach to the dendritic shaft and wrap around a spine, forming synaptic junctions. Electron‐microscopic analyses on the control and afadin‐deficient synapses revealed that afadin plays multiple roles in its ultrastructural morphogenesis of this complex structure.
Information about mechanical reinforcement of decorative organs could help development of a novel technique that would give flowers with robust floral organs and broadly contribute to postharvest ...flower preservation. Hydrangeas (Hydrangea spp.) exhibit remarkable characteristics in terms of mechanical reinforcement of decorative sepals. Although decorative sepals at the flowering stage shrink when they are desiccated, decorative sepals after flowering maintain their shape even after desiccation. In this study, the lignifications of the vein cells in decorative sepals were analyzed using phloroglucinol/HCl-staining. The microstructure of the cell wall was analyzed using transmission electron microscopy (TEM). The three-dimensional structure of vein cells was analyzed using serial block-face scanning electron microscopy (SBF-SEM). Tubular- and spindle-shaped dead cells with a lignified pitted secondary cell wall were observed around the vessel elements in decorative sepals after flowering. These cells were observed as living cells without a secondary cell wall in the veins of decorative sepals at flowering and in fully expanded leaves. Further, 10 hydrangea cultivars were analyzed for development of mechanical reinforcement in vein cells, and some of them were compared by desiccation testing. Decorative sepals of a cultivar lacking those cells exhibited shrinkage after flowering when they were desiccated. In conclusion, dead cells with a lignified pitted secondary cell wall contribute to the reinforcement of veins in decorative sepals of hydrangeas and become sclerified parenchyma cells. Axial parenchyma sclerifying in veins after flowering is essential for robust hydrangea floral organs and represent a new type of mechanical reinforcement tissue in plant decorative floral organs.
PDF
