Tatsuo Ushiki

Scc1/Rad21/Mcd1 Is Required for Sister Chromatid Cohesion and Kinetochore Function in Vertebrate Cells

Proteolytic cleavage of the cohesin subunit Scc1 is a consistent feature of anaphase onset, although temporal differences exist between eukaryotes in cohesin loss from chromosome arms, as distinct from centromeres. We describe the effects of genetic deletion of Scc1 in chicken DT40 cells. Scc1 loss caused premature sister chromatid separation but did not disrupt chromosome condensation. Scc1 mutants showed defective repair of spontaneous and induced DNA damage. Scc1-deficient cells frequently failed to complete metaphase chromosome alignment and showed chromosome segregation defects, suggesting aberrant kinetochore function. Notably, the chromosome passenger INCENP did not localize normally to centromeres, while the constitutive kinetochore proteins CENP-C and CENP-H behaved normally. These results suggest a role for Scc1 in mitotic regulation, along with cohesion.

Elasticity mapping of living fibroblasts by AFM and immunofluorescence observation of the cytoskeleton

Using the force mapping mode of atomic force microscopy (AFM), we measured spatial distribution of elastic moduli of living mouse fibroblasts (NIH3T3) in a physiological condition. The nuclear portion of the cellular surface is about 10 times softer than the surroundings. Stiffer fibers are confirmed in the elastic images. In order to investigate origin of the softer nuclear portion and the stiffer fibers, we fixed the identical cells imaged by the AFM, and carried out immunofluorescence observation for three types of cytoskeletal filaments--actin filaments, microtubules, and intermediate filaments, using confocal laser scanning microscopy (CLSM). A comparison between the AFM and the CLSM images revealed that the elasticity of the cells was concerned not only with the distribution of actin network, but also with intermediate filaments, whereas microtubules had no large effect on the measured elasticity.

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

ABSTRACT Latency-associated nuclear antigen 1 (LANA1) of Kaposis 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 (ΔN-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 ΔN-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.

A scanning electron-microscopic study of the rat thymus with special reference to cell types and migration of lymphocytes into the general circulation.

SummaryThe three-dimensional structure of the rat thymus was studied by combined scanning and transmission electron microscopy. The thymus consists mainly of four types of cells: epithelial cells, lymphocytes, macrophages, and interdigitating cells (IDCs).The epithelial cells form a meshwork in the thymus parenchyma. Cortical epithelial cells are stellate in shape, while the medullary cells comprise two types: stellate and large vacuolated elements. A continuous single layer of epithelial cells separates the parenchyma from connective tissue formations of the capsule, septa and vessels. Surrounding the blood vessels, this epithelial sheath is continuous in the cortex, while it is partly interrupted in the medulla, suggesting that the blood-thymus barrier might function more completely in the cortex.Cortical lymphocytes are round and vary in size, whereas medullary lymphocytes are mainly small, although they vary considerably in surface morphology.Two types of large wandering cells, macrophages and IDCs, could be distinguished, as well as intermediate forms. IDCs sometimes embraced or contacted lymphocytes, suggesting their role in the differentiation of the latter cells.Perivascular channels were present around venules and some arterioles in the cortico-medullary region and in the medulla. A few lymphatic vessels were present in extended perivascular spaces.The present study suggests the possible existence of two routes of passage of lymphocytes into the general circulation. One is via the lymphatics, while the other is through the postcapillary venules into the blood circulation. Our SEM images give evidence that lymphocytes use an intracellular route, i.e., the endothelium of venules.

Structural Changes of Collagen Components and Diminution of Nerves in Congenital Ureteropelvic Junction Obstruction

PURPOSE Three-dimensional arrangements of smooth muscle cells, collagenous component and peripheral nerves of congenital ureteropelvic junction (UPJ) obstruction were studied in order to clarify the pathogenetic mechanism of interaction among these neuro-myo-stromal components. MATERIALS AND METHODS The UPJ and upper ureters were obtained from 14 patients with congenital hydronephrosis (7 intrinsic and 4 extrinsic obstruction) and 7 normal controls. Three-dimensional arrangement of each structural component was observed by scanning electron microscopy, and the nerve distribution was analyzed with immunohistochemistry for protein gene product 9.5. RESULTS The UPJ of intrinsic obstruction had structural features as follows. Muscle fascicles were sparse and thin. Each muscle cell was thin in diameter. Intercellular spaces were six to seven times wider than controls. Collagen fibrillar sheaths of smooth muscle cells (pericellular collagen fibrils attached to the basement membrane) were interwoven to form a dense felt-like structure against thin lace-like sheaths in controls. Interstitial collagenous component showed dense and compact structure against loose network of wavy collagen bundles in controls. In the muscular layer, nerve distribution was decreased to about one-third of controls. In contrast, non-stenotic portion of intrinsic UPJ obstruction as well as materials from extrinsic UPJ obstruction showed no structural difference as compared with controls. CONCLUSIONS In the intrinsic obstruction, nerve fibers were depleted in the muscular layers in the ureteric walls, resulting in dysfunction and atrophy of muscle fibers and an increase of collagen fibers in the muscle layers with abnormal accumulation of intercellular and interstitial collagen. These changes may disrupt the mobility of UPJ and lead to both mechanical and functional obstruction.

Three-Dimensional Arrangement of Collagen and Elastin Fibers in the Human Urinary Bladder: A Scanning Electron Microscopic Study*

To clarify the arrangements of collagen and elastin fibers of the urinary bladder, we examined 9 human (male, aged 42 to 72) urinary bladders by scanning electron microscopy with chemical digestion methods. The mucosal layer was divided into 3 portions according to the collagen arrangement: the superficial portion interwoven densely by collagen fibrils, the middle portion layered by flat bundles of collagen fibrils and the deep portion made of a loose network of twisted collagen bundles. In the muscular layer, the smooth muscle fascicles were firmly covered with collagen sheets, while each muscle cell in a fascicle was accommodated by a thin sheath of collagen fibrils. The serosal layer consists of wavy collagen bundles piled up in a sheet, which was intercalated by clusters of adipose cells. Elastic fibers were, on the other hand, sparse throughout the bladder wall, except for denser networks around the blood vessels and muscle fascicles and beneath the peritoneal mesothelium. The arrangements of these components were discussed in relation to the mechanical function and compliance of the urinary bladder.

Long pentraxin 3 (PTX3) expression and release by neutrophils in vitro and in ulcerative colitis

Pentraxin 3 (PTX3) is the first identified long pentraxin, and it is rapidly produced and released by several cell types in response to proinflammatory signals. The aim of this study was to investigate the behavior of neutrophils to produce PTX3 protein in response to proinflammatory cytokine IL‐8 in vitro, as well as identify the expression pattern of PTX3 in human ulcerative colitis lesions.

Three-dimensional characterization of interior structures of exocytotic apertures of nerve cells using atomic force microscopy

We examined the interior structure of exocytotic apertures in synaptic vesicles of neuroblastoma x glioma hybrid cells using atomic force microscopy. The atomic force microscopy detected apertures of 50-100nm in diameter at various depths within the varicosities of these cells. We were also able to image a regular radial pattern on the wall and lump-like structures at the bottom of these apertures. In contrast, scanning electron microscopy could only detect the apertures but not the fine details of their interior. The cells examined here exhibited the same electrophysiological properties and expression of synaptophysin and syntaxin 1 as presynaptic terminals, as studied by various electrophysiological and imaging techniques. Our results indicate that atomic force microscopy allows three-dimensional viewing of the fine structures located inside exocytotic apertures in nerve cells.

Myocardium Extending from the Left Atrium onto the Pulmonary Veins: A Comparison Between Subjects with and Without Atrial Fibrillation

TAGAWA, M., et al.: Myocardium Extending from the Left Atrium onto the Pulmonary Veins: A Comparison Between Subjects with and Without Atrial Fibrillation. Rapid discharges from the myocardium extending from the left atrium onto the pulmonary vein (PV) have been shown to initiate AF, and AF may be eradicated by the catheter ablation within the PV. However, if there is any difference in the distribution patterns of the myocardial sleeve onto the PV between the subjects with and without AF is to be determined. Twenty‐one autopsied hearts were examined. Eleven patients previously had AF before death and another 10 patients had normal sinus rhythm as confirmed from the medical records including ECGs before death. After exposing the heart, the distance to the peripheral end of the myocardium was measured from the PV‐atrial junction in each PV. Then, the PVs were sectioned and stained and the distal end of myocardium and the distribution pattern were studied. The anteroposterior diameter of the left atrium was also measured. In 74 of 84 PVs, the myocardium extended beyond the PV‐atrial junction. The myocardium was localized surrounding the vascular smooth muscle layer forming a myocardial sleeve. The peripheral end of the myocardial sleeve was irregular and the maximal and minimal distances were measured in each PV. The myocardium extended most distally in the superior PVs compared to the inferior ones and the maximal distance to the peripheral end was similar between the AF and non‐AF subjects (8.4 ± 2.8 vs 8.7 ± 4.4 mm for the left superior and 6.5 ± 3.5 vs 5.1 ± 3.9 mm for the right superior PV, respectively). A significant difference was found in the maximal distance in the inferior PVs: 7.3 ± 4.6 vs 3.3 ± 2.8 mm for the left (P < 0.05) and 5.7 ± 2.4 vs 1.7 ± 1.9 mm for the right inferior PV (P < 0.001) in the subjects with and without AF, respectively. The diameter of left atrium was slightly dilated in AF patients but insignificantly (4.1 ± 0.1 vs 3.6 ± 0.1 cm, P > 0.07). The myocytes on the PV were less uniform and surrounded by more fibrosis in patients with AF compared to those without AF. In conclusion, the myocardium extended beyond the atrium‐vein junction onto the PVs. The distribution patterns of the myocardium was almost similar between subjects with and without AF, but the histology suggested variable myocytes in size and fibrosis in patients with AF.

The olfactory route for cerebrospinal fluid drainage into the peripheral lymphatic system

Drainage of the cerebrospinal fluid through the olfactory nerves into the nasal lymphatics has been suggested repeatedly. To investigate precisely the morphology of this pathway, India ink was injected into the subarachnoidal space of the rat brain, and samples including the olfactory bulbs, olfactory tracts and the nasal mucosa were observed by light and electron microscopy. Under the dissecting microscope, ink particles were found within the subarachnoid space and along the olfactory nerves. At the nasal mucosa, a lymphatic network stained in black was identified near the olfactory nerves, which finally emptied into the superficial and deep cervical lymph nodes. Light microscopically, ink particles were found in the subarachnoid space, partially distributed around the olfactory nerves and within the lymphatic vessels. By electron microscopy, the subarachnoid space often formed a pocket‐like space in the entrance of the fila olfactoria. The olfactory nerves were partially surrounded by ink particles within the space between perineurial cells and epineurial fibroblasts. At the nasal mucosa, the lymphatics were frequently located close to the nerves. These results indicate that the cerebrospinal fluid drains from the subarachnoid space along the olfactory nerves to the nasal lymphatics, which in turn, empties into the cervical lymph nodes. This anatomical communication, thus, allows the central nervous system to connect with the lymphatic system. The presence of this route may play an important role in the movement of antigens from the subarachnoidal space to the extracranial lymphatic vessels, resulting in inducement of an immune response of the central nervous system.