Language：English   Publishing type：Research paper (scientific journal)   Publisher：SPRINGER  

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.

DOI： 10.1007/s10577-008-1241-7

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：BLACKWELL PUBLISHING  

Background: We have investigated how recirculating lymphocytes patrol the liver in a normal steady state. Methods: Thoracic duct lymphocytes of congeneic rats were intravenously transferred to host rats and donor cell trafficking in the liver and hepatic lymph was examined. Host hepatic lymph nodes (HLNs) were selectively removed, which allowed liver-derived donor cells to collect in the thoracic duct without transit in the intervening HLNs. Results: The number of donor cells in the thoracic duct lymph significantly increased over the baseline 3, 5 and 11 h after transfer in the HLN-removed, non-pretreated, and HLN-ligated ( in which a lymph efflux was blocked) groups, respectively. Histologically, donor cells appeared in the portal area from 0.5 h after transfer and frequently attached to the basal lamina of portal vein both externally and internally. Three hours after transfer, a few donor cells appeared in the subcapsular sinus of HLNs. Conclusion: The minimal transit time of rat recirculating lymphocytes is 3 - 4 h in the liver and 5 - 8 h in the hepatic LNs, in a normal steady state. Recirculating lymphocytes might transmigrate through the portal vein as well as the sinusoid in the periportal zone. This rapid transit might enable an efficient surveillance of the liver portal area by the recirculating lymphocytes.

DOI： 10.1111/j.1478-3231.2008.01671.x

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Language：English   Publisher：BIOPHYSICAL SOCIETY  

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：INT SOC HISTOLOGY & CYTOLOGY  

The arrangement of connective tissue components (i.e., collagen, reticular, and elastic fibers) and glial elements in the optic nerve head of the human eye was investigated by the combined use of light microscopy and scanning electron microscopy (SEM). Light-microscopically, the optic nerve head could be subdivided into four parts from the different arrangements of the connective tissue framework: a surface nerve fiber layer, and prelaminar, laminar, and postlaminar regions. The surface nerve fiber layer only possessed connective tissue elements around blood vessels. In the prelaminar region, collagen fibrils, together with delicate elastic fibers, formed thin interrupted sheaths for accommodating small nerve bundles. Immunohistochemistry for the glial fibrillary acidic protein (GFAP) showed that GFAP-positive cells formed columnar structures (i.e., glial columns), with round cell bodies piled up into layers. These glial columns were located in the fibrous sheaths of collagen fibrils and elastic fibers. In the laminar region, collagen fibrils and elastic fibers ran transversely to the optic nerve axis to form a thick membranous layer - the lamina cribrosa - which had numerous round openings for accommodating optic nerve fiber bundles. GFAP-positive cellular processes also ran transversely in association with collagen and elastin components. The postlaminar region had connective tissues which linked the lamina cribrosa with fibrous sheaths for accommodating nerve bundles in the extraocular optic nerve, where GFAP-positive cells acquired characteristics typical of fibrous astrocytes. These findings indicate that collagen fibrils, as a whole, form a continuous network which serves as a skeletal framework of the optic nerve head for protecting optic nerve fibers from mechanical stress as well as for sustaining blood vessels in the optic nerve. The lamina cribrosa containing elastic fibers are considered to be plastic against the mechanical force affected by elevation of the intraocular pressure. The present study has also indicated that glial cells with an astrocytic character play an important role in constructing the connective tissue framework characteristic of the optic nerve head.

DOI： 10.1679/aohc.69.341

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：INT SOC HISTOLOGY & CYTOLOGY  

The three-dimensional ultrastructure of the Golgi apparatus in different cells of the rat - epithelial principal cells in the epididymal duct, goblet cells in the jejunum, gonadotrophs in the pituitary grand and dorsal root ganglion cells - was studied by scanning electron microscopy (SEM) of osmium-macerated tissues. The Golgi apparatus in the epididymal principal cells took the shape of a candle flame with irregular-shaped cisterns, while those in the goblet cells of the jejunum were cup-shaped or cylindrical with flat cisterns. Gonadotrophs had a large spherical Golgi apparatus; this apparatus was composed of several concentric cisterns with large round windows through which the rough endoplasmic reticulum (rER) and mitochondria extended into the center of the globular Golgi apparatus. Dorsal root ganglion cells had several small Golgi stacks scattered in the cytoplasm. In all Golgi apparatuses of the different cells examined in the present study, the cis-most cistern was generally composed of a flattened sheet with numerous small fenestrations on its wall. On the other hand, the shape of the trans-most cistern varied by cell type; it was generally composed of tubules and/or small sheets which were sometimes connected with each other to form a rather complicated structure. The cismost cistern and the trans-most cistern were often closely associated with the rER although no direct communication was found between them. These findings indicate that the structure of the Golgi apparatus, especially its overall shape and the ultrastructure of the trans-most cistern, varies by cell type, a point to be considered in relation to the function of the individual cells.

DOI： 10.1679/aohc.69.357

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：AMER SOC BIOCHEMISTRY MOLECULAR BIOLOGY INC  

Immunoglobulin heavy-chain (IgH) class switch recombination (CSR) is initiated by DNA breakage in the switch (S) region featuring tandem repetitive nucleotide sequences. Various studies have demonstrated that S-region transcription and splicing proceed to genomic recombination and are indispensable for CSR in vivo, although the precise molecular mechanism is largely unknown. Here, we show the novel physical property of the in vitro transcribed S-region RNA by direct visualization using an atomic force microscope (AFM). The S-region sense RNA, but not the antisense RNA, forms a persistent hybrid with the template plasmid DNA and changes the plasmid conformation from supercoil to open circle in the presence of spermidine. In addition, the S-region transcripts generate globular forms and are assembled on the template DNA into a large aggregate that may stall replication and increase the recombinogenicity of the S-region DNA.

DOI： 10.1074/jbc.M209262200

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：AMER SOC MICROBIOLOGY  

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.

DOI： 10.1128/JVI.76.24.12917-12924.2002

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：ELSEVIER SCIENCE INC  

Purpose: We attempted to analyze the three-dimensional ultrastructure of human corneal and scleral collagen fibrils with an atomic force microscope (AFNI).
Methods: A normal eye removed from a 66-year-old male patient during therapy was used in this study. Suspended corneal and scleral collagen fibrils were individually attached to glass slides by centrifugation. These collagen fibrils were air-dried and observed with a non-contact-mode AFM in air.
Results: AFM imaging provided information on the surface topography of both corneal and scleral collagen fibrils. The corneal collagen fibrils had a height of 11.9 +/- 1.0 nm (mean +/- SD) and scleral fibrils a height of 82.5 +/- 35.6 nm. A periodic banding pattern of grooves and ridges was clearly found on both types of fibrils; the D-periodicity and the groove depth were 65.7 +/- 0.8 nm and 1.46 +/- 0.50 nm in the corneal fibrils, and 67.3 +/- 1.1 nm and 6.16 +/- 1.23 nm in the scleral fibrils.
Conclusions: Surface topographic images of human corneal and scleral collagen fibrils were clearly obtained with the AFM. This technique provides quantitative information on the surface morphology of the collagen fibrils at high resolution. (C) 2002 Japanese Ophthalmological Society.

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Language：English   Publisher：INT SOC HISTOLOGY & CYTOLOGY  

Fibrous components of the extracellular matrix are light-microscopically classified into three types of fibers: collagen, reticular and elastic. The present study reviews the ultrastructure of these fibrous components as based on our previous studies by light, electron, and atomic force microscopy.
Collagen fibers present a cord- or tape-shape 1-20 mum wide and run a wavy course in tissues. These fibers consist of closely packed thin collagen fibrils (30-100 nm thick in ordinary tissues of mammals), and exhibit splitting and joining in altering the number of the fibrils to form a three-dimensional network as a whole. Individual collagen fibrils (i.e., unit fibrils) in collagen fibers have a characteristic D-banding pattern whose length is ranges from 64 to 67 nm, depending on tissues and organs. During fibrogenesis, collagen fibrils are considered to be produced by fusing short and thin fibrils with tapered ends.
Reticular fibers are usually observed as a delicate meshwork of fine fibrils stained black by the silver impregnation method. They usually underlie the epithelium and cover the surface of such cells of muscle cells, adipose cells and Schwann cells. Electron-microscopically, reticular fibers are observed as individual collagen fibrils or a small bundle of the fibrils, although the diameter of the fibrils is thin (about 30 nm) and uniform. Reticular fibers are continuous with collagen fibers through the exchange of these collagen fibrils. In silver-impregnated specimens, individual fibrils in reticular fibers are densely coated with coarse metal particles, probably due to the high content of glycoproteins around the fibrils.
Elastic fibers and laminae are composed of microfibrils and elastin components. Observations of the extracted elastin have revealed that elastin components are comprised of elastin fibrils about 0.1-0.2 mum thick. Elastic fibers and laminae are continuous with networks and/or bundles of microfibrils (or oxytalan fibers), and form an elastic network specific to individual tissues.
The fibrous components of the extracellular matrix are thereby morphologically categorized into two systems: the collagen fibrillar system as a supporting framework of tissues and cells, and the microfibril-elastin system for uniformly distributing stress to maintain the resilience adapted to local tissue requirements.

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：INT SOC HISTOLOGY & CYTOLOGY  

Structural changes in the developing rat lung were studied by a combined use of light microscopy including immunohistochemistry for alpha-smooth muscle actin (alpha-SMA) and scanning electron microscopy (SEM) using the KOH-collagenase digestion method. In the embryonic stage (E11-E13), the lung bud appeared as an outgrowth from the ventral wall of the foregut which grew caudally into the splanchnic mesoderm to form a pair of bronchial buds at the end. At E13, the airway smooth muscle cells first appeared around the bifurcation of the trachea. These smooth muscle cells were restricted to the dorsal surface of the tracheal epithelium, suggesting a difference in character between the dorsal and ventral sides of the mesenchymal cells in this region. During the pseudoglandular stage (E13-E18.5), the bronchial buds repeatedly gave off branches in the mesenchymal tissue. The smooth muscle cells in the bronchioles were spindle-shaped and arranged completely circularly around the epithelial tube, except that the terminal bud of bronchioles lacked the smooth muscles. The neck of the terminal bud was constantly surrounded by flat and irregularly-shaped immature smooth muscle cells, representing an early event in the smooth muscle cell differentiation from mesenchymal cells. In the canalicular to saccular stages (E18.5 to birth), the terminals of bronchioles became saccular, thus forming prospective alveolar acini. At birth, the alveolar wall became thinner than before birth, and the individual smooth muscle cells in bronchioles were elongated like a tape. As to the blood vessel differentiation, various sized sinusoidal spaces indicating the primitive blood vessels were already present in the mesenchymal tissue at E11.5. The endothelial cells of these sinusoidal spaces were irregularly shaped and sometimes extended their processes into the lumen. The network of tubular vessels appeared from E14.5. These vessels had tapering ends as well as transluminal trabeculae, suggesting that capillary growth proceeds by both the sprouting and partitioning (i.e., intussusception) of vessels in the pseudoglandular stage.

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：INT SOC HISTOLOGY & CYTOLOGY  

The hepatic portal vein has been known to make a spontaneous peristaltic movement in some mammals, including the mouse and rat. To investigate the fine structure of the portal vein in relation to its physiological characteristics, we observed the mouse portal vein by using various histological techniques including conventional light microscopy, videomicroscopy, transmission and scanning electron microscopy, and real-time confocal laser scanning microscopy.
The mouse hepatic portal vein was provided with a spiral fold which was produced by the inner layer, i.e. the endothelium and smooth muscles of the wall protruding into the lumen. Longitudinal smooth muscle cells spanned the interval of the fold, like a spirally arranged palisade around the vessel wall.
The longitudinal muscle fibers ended at the spiral fold, being partly connected with a network of irregularly shaped smooth muscle cells. This network, hitherto unknown, was recognized to be restricted to the fold in distribution and characterized by numerous gap junctions connecting the muscle cells.
Real-time confocal laser scanning microscopy using a Ca2+ sensitive fluorescent dye revealed that a transient and periodic increase in Ca2+ concentration occurred in the longitudinal smooth muscle cells and was transmitted spirally from the intestinal to the hepatic side. These findings indicate that, during the peristaltic movement, the contraction of smooth muscle cells is transmitted along the longitudinal smooth muscles of the portal vein wall toward the liver, presumably controlled by the network of the irregularly-shaped smooth muscle cells in the fold of the portal vein.
Light microscopic observation in some specimens indicated an occurrence of cardiac muscle cells outside the smooth muscle layer. Restricted to the site of the porta hepatis in distribution, their involvement in the peristaltic contraction of the portal vein seemed unlikely.

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：OXFORD UNIV PRESS  

The present study investigated the applicability of scanning near-field optical/atomic force microscopy (SNOM/AFM) to observations of YOYO-1 stained barley chromosomes. Using this technique, topographic and fluorescent images in the same portion were obtained simultaneously, thus enabling the precise analysis of fluorescent structures in relation to the morphology of chromosomes. In YOYO-1 stained chromosomes, the fluorescent intensity roughly reflected the local amount and/or density of DNA in chromosomes. R-banding of chromosomes stained with YOYO-1 and methyl green was also observed clearly as bright spots of various sizes by this microscope. Thus, the SNOM/AFM is expected to become a useful tool for analysing the structure and function of chromosomes more precisely than before.

DOI： 10.1093/jmicro/51.3.199

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