Osamu Hoshi

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.

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.

Morphological analysis of acute ulcerative colitis experimentally induced by dextran sulfate sodium in the guinea pig: Some possible mechanisms of cecal ulceration

This study in the guinea pig demonstrated that ulcerative colitis-like lesions were induced more rapidly and effectively than in other laboratory animals by the oral administration of dextran sulfate sodium (DSS). In all guinea pigs receiving 3% DSS solution, diarrhea was noted within 48 h, and bleeding at 48–72 h. Light microscopically, hemorrhagic and ulcerative lesions were observed in the cecum and proximal colon as early as 72 h after administration. Lamina propria macrophages, gather in the subepithelial region under normal conditions, were markedly increased in number after DSS administration; by 96 h, they were approximately three times as many as in the control specimens. The cecal mucosa was also characterized, in earlier stages, by the obliteration of virtually all cryptal lumina because of the accumulation of mucous secretions, leading to the subsequent disappearance of the crypts. The obliteration of crypts, which preceded the increase of macrophages, is suggested to play a leading part in this ulceration.

High-speed AFM of human chromosomes in liquid

Further developments of the previously reported high-speed contact-mode AFM are described. The technique is applied to the imaging of human chromosomes at video rate both in air and in water. These are the largest structures to have been imaged with high-speed AFM and the first imaging in liquid to be reported. A possible mechanism that allows such high-speed contact-mode imaging without significant damage to the sample is discussed in the context of the velocity dependence of the measured lateral force on the AFM tip.

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.

Simultaneous activation of natural killer T cells and autoantibody production in mice injected with denatured syngeneic liver tissue

Denatured syngeneic liver tissue prepared by mechanical procedures was intraperitoneally injected into adult C57BL/6 mice. In parallel with a decrease in the total number of lymphocytes in the liver, spleen, and thymus from days 1–7 after the injection, the proportion of the CD4+NK1·1+CD3int subset of these cells (i.e. natural killer T or NKT cells) increased in the liver. Even the absolute number of these NKT cells increased in the liver on days 14 and 21. In response to the injection of denatured liver tissue, tissue damage was induced in the liver, as shown by elevated levels of serum transaminases and hepatocyte degeneration observed by electron microscopy. Sera obtained on days 7 and 14 contained autoantibodies including anti‐DNA antibodies. The proportion of CD1dhighB cells in the liver was found to decrease on days 1–7. In other words, denatured liver tissue stimulated both NKT cells and certain B cells in the liver. These results suggest that liver lymphocytes might contain not only autoreactive T cells (e.g. CD3int or NKT cells) but also some B cells (e.g. B‐1 cells) which produce autoantibodies and that the denatured tissue had the potential to stimulate these lymphocytes and to evoke an autoimmune‐like state.

Selective uptake of intraluminal dextran sulfate sodium and senna by macrophages in the cecal mucosa of the guinea pig

The involvement of macrophages in the passage of intraluminal substances into the lamina propria was examined in the large intestine of the guinea pig. Dextran sulfate sodium (DSS) and senna, which, experimentally, induce ulcerative colitis and melanosis coli, respectively, were chosen for examination, since these substances are visible under the microscope without any special treatment. DSS (MW 50 000) and senna were orally administered to guinea pigs. In tissue sections of the intestine, the presence of DSS was demonstrated by toluidine blue staining, while senna was visible under the light microscope as brown pigment. In the large intestine of guinea pigs, macrophages were most numerous in the cecum, decreasing in number towards the rectum. Metachromatic reaction due to DSS was first recognized in the epithelium of the cecum, and was subsequently incorporated by macrophages. The presence of DSS, either in the epithelium or in macrophages, was not recognized in the small intestine or the distal colon. Senna pigmentation was also limited to the cecum and proximal colon, in which pigmented macrophages aggregated in the lamina propria. The two different substances administered orally were taken up in the cecum, and partly also in the proximal colon; the substances passed through the epithelium and were incorporated by macrophages. This finding suggests the existence of a weak point in the intestinal barrier in this particular portion of the intestine.

Nitric Oxide Synthase in Rat Nasal Mucosa; Immunohistochemical and Histochemical Localization

The localization of nitric oxide synthase (NOS) and its cofactor, nicotinamide-adenine dinucleotide hydrogen phosphate (NADPH)-diaphorase, was examined in the nasal mucosa of the rat by immunohistochemical and histochemical methods. In addition to cryostat sections, whole mount preparations were used to examine the distribution of nerves. Both in the nasal mucosa and in associated ganglia, the distribution of NOS-immunoreactive nervous structures essentially corresponded to that of NADPH-diaphorase-positive ones. The NOS-immunopositive nerve fibers in the respiratory area of the nasal mucosa were distributed around blood vessels and in submucosal glands. Part of the respiratory area was supplied with intraepithelial arborizations of the immunopositive fibers. The epithelial cells in the respiratory area were NADPH-diaphorase positive but NOS immunoreactivity negative. In the olfactory area, the NADPH-diaphorase- and NOS-positive nerve fibers were restricted to blood vessels located deep in the submucosa. Throughout the nasal mucosa, arterial endothelium was NADPH-diaphorase positive but NOS immunoreactivity negative. Both NOS immunoreactivity and NADPH-diaphorase activity were found in major populations of neuronal somata in the sphenopalatine ganglion. The present study provides the direct evidence supporting the notion that nitric oxide is richly produced in autonomic nerves of the nasal mucosa derived from the sphenopalatine ganglion.

Topographical Relation between Serotonin-Containing Paraneurons and Peptidergic Neurons in the Intestine and Urethra

Luminal stimulation of enterochromaffin (EC) cells is known to cause their release of serotonin which in turn may induce a variety of reflexes via mucosal intrinsic neurons. To morphologically support this idea, the present study demonstrates a close topographical relationship between EC cells and nerves, using whole-mount preparations. Beaded nerve fibers containing vasoactive intestinal polypeptide (VIP) were observed in close proximity to serotonin-immunoreactive EC cells in the small and large intestines of the rat and guinea pig; in whole-mount preparations, the VIP nerve fibers appeared to underlie EC cells. This finding correlates with the physiological datum that the intra-arterial infusion of serotonin causes VIP release from the intestine. Canine urethral serotonin cells, a counterpart of the intestinal EC cells, were shown to contact intraepithelial nerves immunoreactive for calcitonin gene-related peptide. The neuroparaneuronal connection in the urethra may play an important role in the serotonin-evoked urethrogenital reflex. Intestinal and urethral serotonin-containing paraneurons, which are sensory in nature, may release serotonin in response to luminal stimuli, and directly activate adjacent peptidergic neurons to initiate the reflex arcs.

Visualization of Elasticity Distribution of Single Human Chromosomes by Scanning Probe Microscopy

We succeeded in visualizing the spatial distribution of the local elasticity of mitotic human chromosomes in a liquid environment using scanning probe microscopy (SPM). Force-versus-indentation curves (force curves) were collected over an entire single chromosome. To estimate the local elasticity of thin chromosomes from the force curves, we examined the validity of a previously proposed model that takes into account the effect of the finite thickness of samples on the estimation of the local elasticity. The force curves obtained are well represented by the model within a small indentation range. The elasticity obtained is independent of the indentation within an indentation range of 100 nm. Such fitting procedures for the force curves collected are carried out over the entire chromosome, and the elasticity distribution of a single chromosome is visualized.