Kiyokazu Kametani

Localization of translational components at the ultramicroscopic level at postsynaptic sites of the rat brain

We investigated the localization of components of translational machinery and their regulators in the postsynaptic region. We examined several components, especially those involved in translational regulation: components of (1) MAPK-Mnk-eIF4E, (2) PI3-kinase-PDK-Akt/PKB-FRAP/mTOR-PHAS/4EBP, (3) p70S6K-S6 ribosomal protein and (4) eEF2 kinase/CaMKIII-eEF2 pathways. Western blotting detected all the components examined in the synaptic fractions, and their differential localization to the synaptic subcompartments: initiation or elongation factors, except for eIF5, were detected predominantly in the dendritic lipid raft fraction, which contained ER marker proteins. In contrast, most of their regulatory kinases were distributed to both the postsynaptic density (PSD) and the dendritic lipid raft fractions, or enriched in the former fraction. Localization of eIF4E at synaptic sites was further examined immunohistochemically at the electron microscopic level. The eIF-4E-immunoreactivity was localized to the postsynaptic sites, especially to the microvesicle-like structures underneath the postsynaptic membrane in the spine, some of which were localized in close proximity to PSD. These results suggest that the postsynaptic local translational system, in at least four major regulatory pathways, is similar to those in the perinuclear one, and that it takes place, at least partly, immediately beneath the postsynaptic membrane. The results also suggest the presence of ER-associated type of translational machinery at the postsynaptic sites.

Human Amniotic Mesenchymal Cells Differentiate into Chondrocytes

Recently, cartilage diseases have been treated by auto- or allogenic chondrocyte transplantation. However, such treatments are limited by the necessity of having a large amount of cells for transplantation, the risk of rejection, and donor shortage. Since the human amnion is immune-privileged tissue suitable for allotransplantation, the potential of human amniotic mesenchymal cells (HAMc) to differentiate into chondrocytes was assessed. The expression of gene encoding transcription factors SOXs and bone morphogenetic proteins (BMPs) as well as BMP receptors were assessed. Chondrocyte phenotype was characterized by positive expression of the cartilage marker genes collagen type II and aggrecan by RT-PCR, collagen type II protein were analyzed by immunofluorescence analysis. HAMc expressed chondrocyte-related genes, including SOXs, BMPs, as well as BMP receptors. Collagen type II and aggrecan were detected after the induction of chondrogenesis with BMP-2. HAMc, transplanted into noncartilage tissue of mice with BMP-2, or implanted with collagen-scaffold into the defects generated in a rats bone, underwent morphological changes with deposition of collagen type II. These results showed that HAMc have the potential to differentiate into chondrocytes in vitro and in vivo, suggesting that they have therapeutic potential for the treatment of damaged or diseased cartilage.

Efferent projections from the striatal patch compartment: anterograde degeneration after selective ablation of neurons expressing μ-opioid receptor in rats

Local injection of mu-opioid receptor specific neurotoxin, dermorphin-saporin, into the striatum resulted in selective degeneration of striatal neurons in the patch compartment. We analyzed subsequent anterograde degeneration of axons and terminals at light and electron microscopic level. Light microscopic examination after silver impregnation method revealed that degenerating axons and terminals arising from the striatal patch compartment were distributed in the globus pallidus, entopeduncular nucleus, and substantia nigra. They were found in both pars reticulata and compacta of the substantia nigra. Electron microscopic examination revealed that the degenerating axon terminals contained large pleomorphic vesicles and formed symmetric synapses on dendrites. The present results suggest that patch neurons expressing mu-opioid receptor send projection fibers to multiple nuclei of the basal ganglia.

A thin carbon-fiber web as a scaffold for bone-tissue regeneration.

Due to the rapid progress being made in tissue regeneration therapy, biomaterials used as scaffolds are expected to play an important role in future clinical application. We report the development of a 3D web (sheet) consisting of high-purity carbon fibers in a nanoscale structure. When the thin carbon-fiber web (TCFW) and recombinant human bone morphogenetic protein 2 (rhBMP-2) composite is implanted in the murine back muscle, new ectopic bone is formed, and the values of the bone mineral content and bone mineral density are significantly higher than those obtained with a collagen sheet. Observation of the interface between the carbon fibers and bone matrix reveal that the fibers are directly integrated into the bone matrix, indicating high bone-tissue compatibility. Further, the rhBMP-2/TCFW composite repairs a critical-size bone defect within a short time period. These results suggest that the TCFW functions as an effective scaffold material and will play an important role in tissue regeneration in the future.

Quantitative elemental analysis on aluminum accumulation by HVTEM-EDX in liver tissues of mice orally administered with aluminum chloride

Quantitative elemental analysis on Al was carried out by high-accelerating voltage transmission electron microscopy (HVTEM) equipped with energy-dispersive X-ray microanalysis (EDX) using an accelerating voltage at 300 kV with high permeability in 1-μm-thick samples obtained from mice administered with aluminum chloride solution for 3, 9, and 17 weeks. By light microscopic observation, no morphological changes were observed in the hepatocytes and macrophages in the liver tissues of mice that were administered with excess Al as compared with the normal control mice. In contrast, by electron microscopic observation, ultrastructural changes were observed in the lysosomes in the hepatocytes as well as the pinocytotic vesicles in the macrophages in the experimental animals. Therefore, the concentrations of Al detected in lysosomes in hepatocytes and pinocytotic vesicles in macrophages of livers of mice administered with Al were measured in relationship to those administration periods. Moreover, transitional changes of hepatocyte lysosome ratios by image analysis and the macrophage counts in the unit area increased in liver tissues of mice administered with Al as compared with normal control mice. From the results, it was demonstrated that hepatocyte lysosome ratio and macrophage count increased in liver tissues of treated mice during those short-term excessive Al administration periods. It was also clarified that the concentrations of Al in both hepatocytes and macrophages increased as observed by HVTEM-EDX. In conclusion, Al accumulated in hepatocytes and macrophages at 3 and 9 weeks administration, while the ultrastructural changes remained in the hepatocytes and macrophages. In contrast, Al concentration did not increase in the liver at 17 weeks administration.

Membrane-associated activation of cholesterol α-glucosyltransferase, an enzyme responsible for biosynthesis of cholesteryl-α-D-glucopyranoside in Helicobacter pylori critical for its survival.

Helicobacter pylori (H. pylori) is the causative pathogen underlying gastric diseases such as chronic gastritis and gastric cancer. Previously, the authors revealed that α1,4-linked N-acetylglucosamine-capped O-glycan (αGlcNAc) found in gland mucin suppresses H. pylori growth and motility by inhibiting catalytic activity of cholesterol α-glucosyltransferase (CHLαGcT), the enzyme responsible for biosynthesis of the major cell wall component cholesteryl-α-d-glucopyranoside (CGL). Here, the authors developed a polyclonal antibody specific for CHLαGcT and then undertook quantitative ultrastructural analysis of the enzyme’s localization in H. pylori. They show that 66.3% of CHLαGcT is detected in the cytoplasm beneath the H. pylori inner membrane, whereas 24.7% is present on the inner membrane. In addition, 2.6%, 5.0%, and 1.4% of the protein were detected in the periplasm, on the outer membrane, and outside microbes, respectively. By using an in vitro CHLαGcT assay with fractionated H. pylori proteins, which were used as an enzyme source for CHLαGcT, the authors demonstrated that the membrane fraction formed CGL, whereas other fractions did not. These data combined together indicate that CHLαGcT is originally synthesized in the cytoplasm of H. pylori as an inactive form and then activated when it is associated with the cell membrane. This article contains online supplemental material at http://www.jhc.org. Please visit this article online to view these materials.

Property analysis of ectopic calcification in the carpal tunnel identification of apatite crystals: a case report

IntroductionA 64-year-old woman presented with symptoms of subacute exacerbation of a year-long carpal tunnel syndrome that was caused by a large calcified mass in the tunnel.ConclusionThe resected mass consisted of very tiny rods, and x-ray diffraction analysis, as well as the component analysis using energy dispersive x-ray microanalysis, revealed the mass to be most compatible with apatite. The back-scattered electron images suggested that precipitation might be a mechanism for development of the calcified mass.

Quantification of Protein A-Gold Staining for Peroxisomal Enzymes by Confocal Laser Scanning Microscopy

The protein A-gold technique has been widely applied for visual localization and quantification of various antigens by electron microscopy. Observation of specimens stained by the protein A-gold technique with conventional light microscopy is difficult because of insufficient sensitivity of the staining. Light microscopic visualization and quantification of the reaction products were attempted employing a confocal laser scanning microscope (CLSM). Liver tissues of normal and peroxisome proliferator-treated rats were fixed and embedded in Lowicryl K4M resin. Ultrathin and thin sections were stained for catalase and a peroxisome-specific β-oxidation enzyme by the protein A-gold technique. Ultrathin sections were observed by electron microscopy and the labeling density for each enzyme was analyzed with an image analyzer. Thin sections were observed with a CLSM in the reflection mode and the intensity of the light reflection was analyzed under the same conditions for all specimens. A comparison of these two observation procedures was also attempted using liver tissues stained with various concentrations of the antibody for catalase. The intensity of the reflection for each, as observed by CLSM, correlated well with the labeling density observed by electron microscopy. CLSM made it possible to quantify and to directly observe protein A-gold staining at the light microscopic level.

X-Ray microanalysis of the secretory granules in goblet cells of mouse intestinal tracts: Changes with age

Changes in the elements in goblet cell secretory granules in three portions of the intestinal tract — duodenum, proximal colon, and distal colon — in mice from 14 days to 18 months after birth were studied by quantitative electron probe X-ray microanalysis on quick-frozen and freeze-dried cryosections by calculating the ratio of each element. In the analysis of five elements, the peaks decreased in the order of sulfur (S), potassium (K), chlorine (Cl), phosphorus (P), and calcium (Ca) in the duodenum at 1 month; in the proximal colon, the peaks declined in the order of K, S, Cl, Ca, and P at 3 months; and in the distal colon, the peaks declined as S, K, Ca, Cl, and P at 3 months. The highest average ratio of S was obtained in the duodenum at 1 month, and this value then declined with age. In the proximal colon, the average ratio of K was the highest at 3 months, then declined afterward with age. The highest average ratio of S was at 3 months, and the highest average ratio of K was at 1 month in the distal colon. The ratio of peak counts to the background was calculated for each type of granule. Significant differences were found in the ratios of S and K between the proximal and distal colon. As for S, the duodenum and the distal colon had the highest ratios and the proximal colon had the second highest. Concerning K, the proximal colon had the highest and the duodenum and the distal colon the second highest ratio. The secretory granules of goblet cells in the three portions of the intestinal tract were shown to contain different amounts of each element. Especially, the amount of S differed most from the proximal to the distal colon.

Ring-Mesh Model of Proteoglycan Glycosaminoglycan Chains in Tendon based on Three-dimensional Reconstruction by Focused Ion Beam Scanning Electron Microscopy

Tendons are composed of collagen fibrils and proteoglycan predominantly consisting of decorin. Decorin is located on the d-band of collagen fibrils, and its glycosaminoglycan (GAG) chains have been observed between collagen fibrils with transmission electron microscopy. GAG chains have been proposed to interact with each other or with collagen fibrils, but its three-dimensional organization remains unclear. In this report, we used focused ion beam scanning electron microscopy to examine the three-dimensional organization of the GAG chain in the Achilles tendon of mature rats embedded in epoxy resin after staining with Cupromeronic blue, which specifically stains GAG chains. We used 250 serial back-scattered electron images of longitudinal sections with a 10-nm interval for reconstruction. Three-dimensional images revealed that GAG chains form a ring mesh-like structure with each ring surrounding a collagen fibril at the d-band and fusing with adjacent rings to form the planar network. This ring mesh model of GAG chains suggests that more than two GAG chains may interact with each other around collagen fibrils, which could provide new insights into the roles of GAG chains.