Ribosomal Protein Dynamics and Its Association with Actin Filaments and Local Translation in Axonal Growth Cones of Dorsal Root Ganglia Neurons.

Local translation in growth cones plays a critical role in responses to extracellular stimuli, such as axon guidance cues. We previously showed that brain-derived neurotrophic factor activates translation and enhances novel protein synthesis through the activation of mammalian target of rapamycin complex 1 signaling in growth cones of dorsal root ganglion neurons. In this study, we focused on 40S ribosomal protein S6 (RPS6), 60S ribosomal protein P0/1/2 (RPP0/1/2), and actin filaments to determine how localization of ribosomal proteins changes with overall protein synthesis induced by neurotrophins. Our quantitative analysis using immunocytochemistry and super-resolution microscopy indicated that RPS6, RPP0/1/2, and actin tend to colocalize in the absence of stimulation, and that these ribosomal proteins tend to dissociate from actin and associate with each other when local protein synthesis is enhanced. We propose that this is because stimulation causes ribosomal subunits to associate with each other to form actively translating ribosomes (polysomes). This study further clarifies the role of cytoskeletal components in local translation in growth cones.

Anatomical study of the bone morphology of the anterior talofibular ligament attachment.

Anterior talofibular ligament (ATFL) injuries are the most common cause of ankle sprains. To ensure anatomically accurate surgery and ultrasound imaging of the ATFL, anatomical knowledge of the bony landmarks around the ATFL attachment to the distal fibula is required. The purpose of the present study was to anatomically investigate the ATFL attachment to the fibula with respect to bone morphology and attachment structures. First, we analyzed 36 feet using microcomputed tomography. After excluding 9 feet for deformities, the remaining 27 feet were used for chemically debrided bone analysis and macroscopic and histological observations. Ten feet of living specimens were observed using ultrasonography. We found that a bony ridge was present at the boundary between the attachments of the ATFL and calcaneofibular ligament (CFL) to the fibula. These two attachments could be distinguished based on a difference in fiber orientation. Histologically, the ATFL was attached to the anterodistal part of the fibula via fibrocartilage anterior to the bony ridge indicating the border with the CFL attachment. Using ultrasonography in living specimens, the bony ridge and hyperechoic fibrillar pattern of the ATFL could be visualized. We established that the bony ridge corresponded to the posterior margin of the ATFL attachment itself. The ridge was obvious, and the superior fibers of the ATFL have directly attached anteriorly to it. This bony ridge could become a valuable and easy-to-use landmark for ultrasound imaging of the ATFL attachment if combined with the identification of the fibrillar pattern of the ATFL.

Anti-DNA antibodies cross-reactive with ß2-glycoprotein I induce monocyte tissue factor through the TLR9 pathway.

Antibodies specific for cardiolipin (CL)-ß2-glycoprotein I (ß2GPI) are known to induce tissue factor (TF) expression by monocytes and endothelial cells which leads to a prothrombotic state in antiphospholipid syndrome (APS), but the mechanism is not fully elucidated. Previously, we reported that the mouse monoclonal anti-CL-ß2GPI antibody WB-6 cross-reacts with DNA, enters monocytes via binding to cell surface DNA, and induces TF expression. The current study aimed to identify the intracellular signaling pathways involved in this process. The binding of WB-6 to CL-ß2GPI or DNA, and endocytosis was not prevented by chloroquine, but pre-treatment of the cells with chloroquine significantly suppressed TF expression. TLR9 inhibitory oligodeoxynucleotide also suppressed the WB-6-induced TF expression, suggesting a pivotal role of the TLR9 pathway in TF production. Serum antibodies obtained from a patient with APS accompanying systemic lupus erythematosus (SLE) bound to both CL-ß2GPI and DNA, and induced TF in normal monocytes. This effect was suppressed by chloroquine, and abolished by removal of the DNA-binding activity. These results suggest that induction of TF expression results from TLR9 activation by DNA which was internalized together with cross-reactive antibodies produced in secondary APS accompanying SLE.

In vitro Neo-Genesis of Tendon/Ligament-Like Tissue by Combination of Mohawk and a Three-Dimensional Cyclic Mechanical Stretch Culture System.

Tendons and ligaments are pivotal connective tissues that tightly connect muscle and bone. In this study, we developed a novel approach to generate tendon/ligament-like tissues with a hierarchical structure, by introducing the tendon/ligament-specific transcription factor Mohawk (MKX) into the mesenchymal stem cell (MSC) line C3H10T1/2 cells, and by applying an improved three-dimensional (3D) cyclic mechanical stretch culture system. In our developed protocol, a combination of stable Mkx expression and cyclic mechanical stretch synergistically affects the structural tendon/ligament-like tissue generation and tendon related gene expression. In a histological analysis of these tendon/ligament-like tissues, an organized extracellular matrix (ECM), containing collagen type III and elastin, was observed. Moreover, we confirmed that Mkx expression and cyclic mechanical stretch, induced the alignment of structural collagen fibril bundles that were deposited in a fibripositor-like manner during the generation of our tendon/ligament-like tissues. Our findings provide new insights for the tendon/ligament biomaterial fields.

BDNF Reduces eEF2 Phosphorylation and Enhances Novel Protein Synthesis in the Growth Cones of Dorsal Root Ganglia Neurons.

The local translation, which is regulated by extracellular stimuli such as guidance molecules, in growth cones of neurons provides a molecular mechanism for axonal development. In this study, we performed immunocytochemistry together with atomic force microscopy to investigate the localization of ribosomal proteins in the growth cones of rat dorsal root ganglion (DRG) neurons. The immunoreactivity of ribosomal protein P0/1/2 and S6, and novel protein synthesis were observed in the central, sterically bulky region of growth cones. Brain derived neurotrophic factor (BDNF) reduced the eEF2 phosphorylation, indicating its activation, and enhanced protein synthesis within 30 min. The effects of BDNF were completely inhibited by rapamycin, an inhibitor of mammalian target of rapamycin (mTOR). These results indicated that BDNF rapidly activates translation and enhances novel protein synthesis in growth cones of DRG though the mTOR signaling.

The expression and localization of RNase and RNase inhibitor in blood cells and vascular endothelial cells in homeostasis of the vascular system.

RNA may be released from vascular cells including endothelial cells in the event of injury and in vascular disease. Extracellular RNAs have been recognized as novel procoagulant and permeability-increasing factors. Extracellular RNA may function as inflammatory host alarm signals that serve to amplify the defense mechanism, but it may provide important links to thrombus formation. Extracellular RNA is degraded by RNase. We propose that RNase and its inhibitor RNase inhibitor (RI) act as modulators of homeostasis in the vasculature to control the functions of extracellular RNA. We aimed to investigate the expression and localization of RNase 1 and RI in cells that contact blood, such as platelets, mononuclear cells, polymorphonuclear cells, and red blood cells. RNase 1 and RI expression and localization in blood cells were compared with those in the human umbilical vein endothelial cell line, EAhy926. Additionally, we further investigated the effect of thrombin on the expression of RNase 1 and RI in platelets. We used an RNase activity assay, reverse transcription-polymerase chain reaction, western blot, immunocytochemistry, transmission electron microscopy, and immunoelectron microscopy (pre- and post-embedding methods). RNase activity in the supernatant from EAhy926 cells was 50 times than in blood cells (after 60 min). RNase 1 mRNA and protein expression in EAhy926 cells was highest among the cells examined. However, RI mRNA and protein expression was similar in most cell types examined. Furthermore, we observed that RNase 1 and von Willebrand factor were partially colocalized in EAhy926 cells and platelets. In conclusion, we propose that high RNase activity is ordinarily released from endothelial cells to support anticoagulation in the vasculature. On the other hand, platelets and leukocytes within thrombi at sites of vascular injury show very low RNase activity, which may support hemostatic thrombus formation. However, activated platelets and leukocytes may accelerate pathologic thrombus formation.

Observation of collagen fibrils produced by osteosarcoma cells using atomic force microscopy.

The present study examined the three-dimensional process of collagen fibril formation in the human osteosarcoma cell line NOS-1 by conventional scanning electron microscopy (SEM) and atomic force microscopy (AFM). SEM images showed collagen fibril formation on the bottom of culture dishes after 1 week of culture. The collagen fibrils had diameters of 30-100 nm. The surfaces of individual fibrils had characteristic grooves and ridges with periodicities of 60-70 nm. AFM images showed that the newly formed collagen fibrils were 30-300 nm in diameter and possessed characteristic grooves and ridges with periodicities of 60-70 nm. The thicker collagen fibrils contained thinner (approximately 30 nm thick) subfibrils that ran in a helical direction along the long axis of the thicker fibrils. Furthermore, twisted structures of collagen fibrils, which possessed a characteristic rope-like structure, were also identified. The ultrastructure of the collagen fibrils was clearly imaged in liquid medium by AFM, and the process of collagen fibril assembly was successfully analyzed under conditions much closer to the physiological state than those afforded by transmission electron microscopy or SEM. AFM also provided a precise morphological measurement, particularly of the vertical distance, of collagen fibrils with nanometer-scale resolution in liquid conditions.

Atomic force microscopy for analyzing metaphase chromosomes: comparison of AFM images with fluorescence labeling images of banding patterns.

The combined use of fluorescence microscopy with atomic force microscopy (AFM) has been introduced to analyze the replication-banding patterns of human chromosomes. Human lymphocytes synchronized with excess thymidine are treated with 5-ethynyl-2'-deoxyuridine (EdU) during the late S phase. EdU-labeled DNA is detected in metaphase chromosomes using Alexa Fluor 488(®) azide, through the 1,3-dipolar cycloaddition reaction of organic azides with the terminal acetylene group of EdU. Chromosomes with EdU incorporated during the late S phase show a banding pattern similar to the G-banding pattern of normal human chromosomes. The comparison between the fluorescence and AFM image of the same chromosome indicates the presence of ridges and grooves in the chromatid arms, which correspond to G-positive and G-negative bands, respectively. This technique of EdU-labeled replication bands combined with AFM is useful to analyze the structure of chromosomes in relation to the banding pattern.

Replication Banding Patterns in Human Chromosomes Detected Using 5-ethynyl-2'-deoxyuridine Incorporation.

A novel technique using the incorporation of 5-ethynyl-2'-deoxyuridine (EdU) into replicating DNA is described for the analysis of replicating banding patterns of human metaphase chromosomes. Human lymphocytes were synchronized with excess thymidine and treated with EdU during the late S phase of the cell cycle. The incorporated EdU was then detected in metaphase chromosomes using Alexa Fluor® 488 azides, through the 1,3-dipolar cycloaddition reaction of organic azides with the terminal acetylene group of EdU. Chromosomes with incorporated EdU showed a banding pattern similar to G-banding of normal human chromosomes. Imaging by atomic force microscopy (AFM) in liquid conditions showed that the structure of the chromosomes was well preserved even after EdU treatment. Comparison between fluorescence microscopy and AFM images of the same chromosome 1 indicated the presence of ridges and grooves in the chromatid arm, features that have been previously reported in relation to G-banding. These results suggest an intimate relationship between EdU-induced replication bands and G- or R-bands in human chromosomes. This technique is thus useful for analyzing the structure of chromosomes in relation to their banding patterns following DNA replication in the S phase.

Atomic force microscopy imaging of human metaphase chromosomes in liquid.

Methods for atomic force microscopy (AFM) imaging of human metaphase chromosomes were -introduced in the present study. Chromosomes from the lymphocytes were fixed and prepared onto glass slides as the chromosome spread, and observed in phosphate-buffered saline by dynamic mode AFM. On the contrary, chromosomes from the human cell line BALL-1 were isolated using the hexylene glycol method, absorbed onto a silane-coated glass slide, and observed in a hexylene glycol buffer solution by dynamic mode AFM. AFM provides three-dimensional topographic images of both fixed and unfixed human chromosomes with height information. The ultrastructural image of a pair of chromatids was also obtained by AFM in a liquid condition. The combined use of the AFM and light microscopy of cytochemically and/or immunocytochemically stained chromosomes is also expected to be useful for studies on the localization of chemical components in relation to the higher-order structure of the chromosomes.

Intermediate structure between chromatin fibers and chromosome revealed by mechanical stretching and SPM measurement.

The morphology of chromosomes (certain rod-shaped structures) is highly reproducible despite the high condensation of chromatin fibers (∼1 mm) into chromosomes (∼1 µm). However, the mechanism underlying the condensation of chromatin fibers into chromosomes is unclear. We assume that investigation of the internal structure of chromosomes will aid in elucidating the condensation process. In order to observe the detailed structure of a chromosome, we stretched a human chromosome by using a micromanipulator and observed its morphology along the stretched region by scanning probe microscopy (SPM). We found that the chromosome consisted of some fibers that were thicker than chromatin fibers. The found fiber was composed of approximately 90-nm-wide beads that were linked linearly. To explore the components of the fiber, we performed immunofluorescence staining of the stretched chromosome. Fluorescence signals of topoisomerase (Topo) IIα, which is known to interact with and support chromatin fibers, and DNA were detected both on the found fiber and beads. Furthermore, after micrococcal nuclease and trypsin treatments, the fibers were found to be mechanically supported by proteins. These results suggest that chromosome comprises an intermediate structure between chromatin fibers and chromosomes.

Structural analysis of human chromosomes by atomic force and light microscopy in relation to the distribution of topoisomerase IIα.

The relationship between the higher-order structure of human metaphase chromosomes and the distribution of topoisomerase IIα was analyzed by a comparison of atomic force microscope (AFM) and fluorescence microscope images of the same chromosome. AFM imaging of chromosomes in liquid revealed the presence of alternating ridges and grooves on the surfaces of the sister chromatids. In contrast, the fluorescence image of the chromosomes stained with the anti-topoisomerase IIα antibody showed that the fluorescence intensity of topoisomerase IIα was not uniform and that there were alternating strong and weak spots along the chromosome axes. A comparison of the AFM image with a fluorescence microscope image of the same chromosome further demonstrated that ridges and grooves corresponded to strong and weak fluorescence intensities of topoisomerase IIα, respectively. These findings suggest that the distribution of topoisomerase IIα has a close connection with the higher-order structure of human metaphase chromosomes.

Atomic force microscopy for imaging human metaphase chromosomes.

The present study introduces the principle of atomic force microscopy (AFM) and reviews our results of human metaphase chromosomes obtained by AFM. AFM imaging of the chromosomes revealed that the chromatid arm was not uniform in structure but had ridges and grooves along its length, which was most prominent in the late metaphase. The arrangement of these ridges and grooves was roughly symmetrical with the counterpart of the paired sister chromatids. AFM imaging of banded chromosomes also showed that the ridges and grooves were related to the G/Q-positive and G/Q-negative bands, respectively. At high magnification, the chromatid was seen to be produced by the compaction of highly twisted chromatin fiber loops, and its compaction tended to be stronger in the ridged regions of the chromosomes than in the grooved regions. Our AFM studies also showed the presence of catenation of chromatin fibers between the ridged portions of the chromatid in the late metaphase. Thus, AFM is useful for obtaining the three-dimensional surface topography not only in ambient conditions but also in physiological liquid conditions, and is expected to be an attractive tool for investigating the structure of chromosomes.

Techniques for imaging human metaphase chromosomes in liquid conditions by atomic force microscopy.

The purpose of this study was to obtain three-dimensional images of wet chromosomes by atomic force microscopy (AFM) in liquid conditions. Human metaphase chromosomes-obtained either by chromosome spreads or by an isolation technique-were observed in a dynamic mode by AFM in a buffer solution. Under suitable operating conditions with a soft triangular cantilever (with the spring constant of 0.08-0.4 N m(-1)), clear images of fixed chromosomes in the chromosome spread were obtained by AFM. For imaging isolated chromosomes with the height of more than 400 nm, a cantilever with a high aspect ratio probing tip was required. The combination of a Q-control system and the sampling intelligent scan (SIS) system in dynamic force mode AFM was useful for obtaining high-quality images of the isolated chromosomes, in which globular or cord-like structures about 50 nm thick were clearly observed on the surface of each chromatid.

Atomic force microscopy of native human metaphase chromosomes in a liquid.

The present study introduces a method for obtaining three-dimensional images of native (i.e., unfixed) chromosomes by atomic force microscopy (AFM) in a liquid. Human metaphase chromosomes were isolated from a human lymphoblast-like cell line, K562, by the hexylene glycol procedure according to Wray and Stubble- field (1970), adsorbed on a silane-coated glass slide, and observed in a dynamic force mode (i.e., intermittent contact mode) of AFM in a hexylene buffer solution. In adequate operating conditions, the shape of chromosomes with paired chromatids was clearly and three-dimensionally observed by AFM. At high magnification, globular or fibrous structures about 50 nm thick could be found on the surface of each chromaid, implying that chromatin fibers were strongly wound or twisted in the chromatid. Thus, AFM imaging enabled the direct visualization of native chromosomes in a liquid at high resolution--which is comparable with that of scanning electron microscopy--and can serve to analyze the mechanism of chromosome condensation and separation in relation to the structure of chromosomes.

Atomic force microscopy of human metaphase chromosomes after differential staining of sister chromatids.

Human metaphase chromosomes, in which 5-bromo-deoxyuridine (BrdU) had been incorporated into the DNA, were treated with the fluorescent plus Giemsa (FPG) method. Use of this method distinctly stained one of the paired sister chromatids with the Giemsa solution due to the difference in content of BrdU in the two chromatids. These chromosomes with their differential staining of sister chromatids were observed by atomic force microscopy (AFM). In the air-dried specimens, one of the paired chromatids that was stained strongly with Giemsa solution was about two times higher than the counterpart that was stained faintly with Giemsa solution. In the critical point dried chromosomes, the height of the Giemsa positive chromatid roughly matched that of the Giemsa negative counterpart. These findings imply that the arrangement of the Giemsa negative chromatid after FPG staining is fragile and easily collapses due to the surface tension of water during air-drying. At higher magnifications, the surface structure differed between Giemsa positive and negative chromatids; the Giemsa positive chromatid (i.e., unifilarly BrdU-incorporated chromatid) was composed of fibrous structures while the Giemsa negative chromatid (i.e., bifilarly BrdU-incorporated chromatid) exhibited a fine granular appearance. These structural changes in the sister chromatids are thought to arise from the ultraviolet irradiation and heating of the chromosomes during FPG staining.

Neutrophil extravasation in rat mesenteric venules induced by the chemotactic peptide N-formyl-methionyl-luecylphenylalanine (fMLP), with special attention to a barrier function of the vascular basal lamina for neutrophil migration.

The present study was performed to investigate morphologically the process of neutrophil extravasation induced by the synthetic bacterial peptide N-formylmethionyl-leucyl-phenylalanine (fMLP) in venules of the rat mesentery by the combined use of intravital microscopy and transmission electron microscopy (TEM). Special attention was given to the interaction of the neutrophils with the endothelial cells and endothelial basal lamina. By intravital microscopy, the rolling and adhesion of leukocytes were observed within 3 min in preparations activated by fMLP. Neutrophils remained in the vascular wall for more than 30 min even after penetration of the endothelium. In this period, neutrophils migrating between endothelial cells and their basal lamina were often observed by TEM. After 40 min application of fMLP, some of the migrating neutrophils parted from the vessel wall into the surrounding connective tissues. There were neutrophils which passed through the small pore of the basal lamina with a cytoplasmic constriction. These findings indicate that the endothelial basal lamina acts as a physical barrier against neutrophil extravasation, thus resulting in the transient retainment of neutrophils beneath the endothelial cells of the venular wall.

Chromosome binding site of latency-associated nuclear antigen of Kaposi's sarcoma-associated herpesvirus is essential for persistent episome maintenance and is functionally replaced by histone H1.

Latency-associated nuclear antigen 1 (LANA1) of Kaposi's sarcoma-associated herpesvirus (KSHV; human herpesvirus 8) persistently maintains a plasmid containing the KSHV latent origin of replication (oriP) as a closed circular episome in dividing cells. In this study, we investigated the involvement of chromosome binding activity of LANA1 in persistent episome maintenance. Deletion of the N-terminal 22 amino acids of LANA1 (DeltaN-LANA) inhibited the interaction with mitotic chromosomes in a human cell line, and the mutant concomitantly lost activity for the long-term episome maintenance of a plasmid containing viral oriP in a human B-cell line. However, a chimera of DeltaN-LANA with histone H1, a cellular chromosome component protein, rescued the association with mitotic chromosomes as well as the long-term episome maintenance of the oriP-containing plasmid. Our results suggest that tethering of KSHV episomes to mitotic chromosomes by LANA1 is crucial in mediating the long-term maintenance of viral episomes in dividing cells.

The structure of human metaphase chromosomes: its histological perspective and new horizons by atomic force microscopy.

Studies on the structure of the human chromosome were reviewed from the histological perspective and discussed in connection with our recent findings obtained mainly by atomic force microscopy (AFM). In this paper, we introduce several hitherto known models of the high-order structure of the metaphase chromosome and discuss the actual structure of chromosomes in relation to such structures as spiral chromatids, chromosome bands, and chromosome scaffolds. In chromosomes treated with Ohnuki's hypotonic solution, the chromosome arms were elongated and showed a characteristic spiral pattern of chromatid fibers. On the other hand, alternating transverse ridges and grooves were clearly observed on the surface of chromosomes treated with 0.025% trypsin for G-banding, and these ridges and grooves corresponded to the dark and pale bands of G-banded chromosomes. Similar findings were also found in chromosomes treated with quinacrine mastards for Q-banding. Fibers bridging the gap between the sister chromatids were often observed in G/Q-banded chromosomes; these fibers tended to be restricted within the G/Q-positive portions, suggesting the presence of chromatin fibers bridging these regions. Based on these findings in conjunction with previous studies, we outlined the high-order structure of the human chromosome. Recent advances in nanotechnology have provided new AFM techniques for the imaging and handling of materials at nano-scale resolution. Application of these techniques to chromosome research is expected to provide valuable information on the chromosome structure in relation to its function.