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Biomedical subjects

Toshio Ohtani

Publications and source records attributed to Toshio Ohtani.

At least 19 recordsLinked to original sources

Chromatin dynamics of unfolding and refolding controlled by the nucleosome repeat length and the linker and core histones.

Chromatin is composed of genomic DNA and histones, forming a hierarchical architecture in the nucleus. The chromatin hierarchy is common among eukaryotes despite different intrinsic properties of the genome. To investigate an effect of the differences in genome organization, chromatin unfolding processes were comparatively analyzed using Schizosaccaromyces pombe, Saccharomyces cerevisiae, and chicken erythrocyte. NaCl titration showed dynamic changes of the chromatin. 400-1000 mM NaCl facilitated beads with approximately 115 nm in diameter in S. pombe chromatin. A similar transition was also observed in S. cerevisiae chromatin. This process did not involve core histone dissociation from the chromatin, and the persistence length after the transition was approximately 26 nm for S. pombe and approximately 28 nm for S. cerevisiae, indicating a salt-induced unfolding to "beads-on-a-string" fibers. Reduced salt concentration recovered the original structure, suggesting that electrostatic interaction would regulate this discrete folding-unfolding process. On the other hand, the linker histone was extracted from chicken chromatin at 400 mM NaCl, and AFM observed the "beads-on-a-string" fibers around a nucleus. Unlike yeast chromatin, therefore, this unfolding was irreversible because of linker histone dissociation. These results indicate that the chromatin unfolding and refolding depend on the presence and absence of the linker histone, and the length of the linker DNA.

Animals↗

Comparative structural biology of the genome: nano-scale imaging of single nucleus from different kingdoms reveals the common physicochemical property of chromatin with a 40 nm structural unit.

Genome function is closely linked to the higher-order chromatin structures. To reveal a structural basis for the interphase chromatin organization, the 'on-substrate' lysis procedure was applied to nuclei isolated from human HeLa cells, chicken erythrocyte cells and yeast Schizosaccharomyces pombe, which possessed different intrinsic properties of the genomes such as histone composition and inter-nucleosomal distance. The isolated nuclei on a coverslip were successively treated with a detergent and a high-salt solution to extract the nuclear membrane and the nucleoplasm, and therefore, atomic force microscopy (AFM) visualized the structural changes in response to the lysis procedure. After the nucleoplasm was extracted, AFM clarified that chromatin fibers, approximately 40 nm in width, were partially released out of the nuclei and that the other chromatin still remaining in the nuclei was composed of granular structures with diameter of 80-100 nm. Thus, these results suggest that the approximately 40 nm fiber would be a stable structural unit and fold the 80-100 nm granules into a one-step higher unit. A common mechanism could be implied regardless of the intrinsic properties of the eukaryotic genomes.

Animals↗

Cyclophosphamide enhances TNF-alpha-induced apoptotic cell death in murine vascular endothelial cell.

Cyclophosphamide (CPA) is one of the therapeutic agents for systemic inflammatory disorders. In murine dermal endothelial cells (F-2), 4-hydroxycyclophosphamide (4-HC), which is active metabolite of CPA, enhanced TNF-alpha-induced DNA fragmentation. In addition, 4-HC was shown to elevate TNF-alpha-induced caspase-3 activation. Caspase-8 activation was identified by the treatment of TNF-alpha, whereas 4-HC was no effect. In contrast, only when treated with 4-HC, caspase-9 activation and the increase in the intracellular expression of Bax were detected. These results suggest that CPA may sensitize endothelial cells to TNF-alpha-induced apoptosis through a mitochondria-dependent pathway and clinically may contribute to the limitation of inflammatory process.

Animals↗

Effects of alpha-hydroxy acids on the human skin of Japanese subjects: the rationale for chemical peeling.

Alpha-hydroxy acid (AHA) agents, such as glycolic acid and lactic acid, have been used as therapeutic agents for more than a quarter of a century. Recently, they have been used as agents to rejuvenate photo-aged skin. It is believed that these AHA agents induce the epidermis to remodel and accelerate desquamation, thus exerting their therapeutic effects. In this study, we investigated the histological differences in skin treated with glycolic, lactic, citric and acetic acids once daily for 6 weeks. The melanin pigments in the basal layer were less prominent in the glycolic and lactic acid-treated skin than in the citric and acetic acid-treated skin. The melanin deposits in the horny layers were equal for all AHA. However, the melanin deposits in the squamous layers were less prominent in the glycolic and lactic acid-treated skins than in the citric and acetic acid-treated skins; this was analogous to observations of the basal layers. Collagen I and procollagen I were increased after treatment with glycolic, lactic and citric acid in the upper dermis, but were not increased with acetic acid treatment. However, the staining of the epidermis and dermis for matrix metalloproteinase-1 (MMP-1) after treatment was not significantly different among the agents. Our data suggest that longer treatment intervals with glycolic and lactic acid can cause improvements in both the epidermal and dermal components and support the usefulness of AHA for rejuvenating photo-damaged skin.

Acetic Acid↗

Recessive dystrophic epidermolysis bullosa: case of non-Hallopeau-Siemens variant with premature termination codons in both alleles.

Dystrophic epidermolysis bullosa (DEB) is caused by mutations in the COL7A1 gene encoding collagen, the major component of anchoring fibrils. Premature termination codon (PTC) mutations in both alleles usually lead to the Hallopeau-Siemens variant that shows the most severe phenotype. We experienced a case of the non-Hallopeau-Siemens variant (nHS-RDEB), which had a mild clinical severity although it has PTC mutations in both alleles. Our patient was a compound heterozygote for a nonsense mutation (R669X) in exon 15 and a nonsense mutation (E2857X) in exon 116. But we confirmed the existence of some anchoring fibrils on electron micrograph. This suggested that a PTC close to the 3' end of COL7A1 does not completely abolish the collagen VII mRNA. We hypothesized that the truncated procollagen VII from the mutant allele with a nonsense mutation (E2857X) in exon 116 included two out of eight cysteines needed for disulfide bond formation, and hence a few functional anchoring fibrils could be formed.

Adult↗

Fabricating and aligning pi-conjugated polymer-functionalized DNA nanowires: atomic force microscopic and scanning near-field optical microscopic studies.

We report a simple method to functionalize DNA with pi-conjugated polymer, forming highly aligned and integrated arrays of pi-conjugated polymer nanowires of a few nanometers diameter. pi-conjugated polymer, polyphenazasiline, having alkylammonium salts on the N atom (PPhenaz-TMA), synthesized in this study can be directly attached to DNA, which can be organized along stretched and aligned DNA molecules on surfaces as a template. Furthermore, PPhenaz-TMA/DNA nanowires were stretched and aligned on surfaces, even when PPhenaz-TMA/DNA complexes formed in solutions. The resulting PPhenaz-TMA/DNA nanowires could be easily converted to oxidized states or metallic nanowires by using adequate oxidant or metal salts. The direct visualization of PPhenaz-TMA/DNA nanowires and its structural changes have been studied by atomic force microscopy and scanning near-field optical microscopy.

DNA↗

Difference in bacterial motion between forward and backward swimming caused by the wall effect.

A bacterial cell that has a single polar flagellum alternately repeats forward swimming, in which the flagellum pushes the cell body, and backward swimming, in which the flagellum pulls the cell body. We have reported that the backward swimming speeds of Vibrio alginolyticus are on average greater than the forward swimming speeds. In this study, we quantitatively measured the shape of the trajectory as well as the swimming speed. The trajectory shape in the forward mode was almost straight, whereas that in the backward mode was curved. The same parameters were measured at different distances from a surface. The difference in the motion characteristics between swimming modes was significant when a cell swam near a surface. In contrast, the difference was indistinguishable when a cell swam >60 microm away from any surfaces. In addition, a cell in backward mode tended to stay near the surface longer than a cell in forward mode. This wall effect on the bacterial motion was independent of chemical modification of the glass surface. The macroscopic behavior is numerically simulated on the basis of experimental results and the significance of the phenomenon reported here is discussed.

Bacterial Physiological Phenomena↗

Isolation and characterization of simple repeat sequences from the yellow fin sea bream Acanthopagrus latus (Sparidae).

We isolated DNA fragments containing various repetitive elements from the genome of a sea bream Acanthopagrus latus. Sequence analysis indicated that two fragments have particularly interesting features. Fragment AL87 contained a tetranucleotide repeat and a quasipalindromic sequence. Sequence comparison suggested that AL87 may be a part of a gene encoding a serine/threonine protein kinase, and that the quasipalindrome is situated at the junction of an intron and an exon. Moreover, the quasipalindrome is conserved in several other fishes, even though it has the potential to form a stem-loop structure at the splicing site. Fragment AL79 contained a minisatellite sequence made up of six 30-bp units in tandem. DNase I sensitivity assays and statistical analyses showed the repeat region to be flexible when subjected to bending stress. In addition, atomic force microscopic imaging of AL79 showed the presence of highly curved (kinked) segments flanking the repeat region. The structural features of these repetitive elements may be key factors facilitating the amplification of the repeats.

Animals↗

Effects of acetic acid treatment on plant chromosome structures analyzed by atomic force microscopy.

Acetic acid treatment has been frequently used to remove cellular contaminants from plant chromosome samples for structural analyses by scanning electron microscopy and atomic force microscopy (AFM). We evaluated the effects of various concentrations of acetic acid treatments on barley chromosome structures by using AFM. The long-term 45% acetic acid treatment significantly damaged the chromosome structures, although the treatment effectively removed the cellular contaminants. On the other hand, the treatment with 15% acetic acid could not obtain sufficiently clean chromosome samples and the chromosome surface structures could not be observed. In contrast, we obtained clean chromosome preparation without severe damage by using an intermediate concentration (30%) of acetic acid treatment. In the centromeric region, we could observe fiber structures with a width of 100 nm, which were composed of ca. 50-nm granules and aligned to the axes of chromosomes. Thus, AFM analysis of chromosomes appropriately treated with acetic acid will provide important insights into the organization of higher-order structures of plant chromosomes.

Acetic Acid↗

Scanning Near-field Optical/Atomic Force Microscopy detection of fluorescence in situ hybridization signals beyond the optical limit.

Fluorescence in situ hybridization (FISH) is widely used in molecular biological study. However, high-resolution analysis of fluorescent signals is theoretically limited by the 300-nm resolution optical limit of light microscopy. As an alternative to detection by light microscopy, we used Scanning Near-field Optical/Atomic Force Microscopy (SNOM/AFM), which can simultaneously obtain topographic and fluorescent images with nanometer-scale resolution. In this study, we demonstrated high-resolution SNOM/AFM imaging of barley chromosome (Hordeum vulgare, cv. Minorimugi) FISH signals using telomeric DNA probes. Besides detecting the granular structures on chromosomes in a topographic image, we clearly detected fluorescent signals in telomeric regions with low-magnification imaging. The high-resolution analysis suggested that one of the telomeric signals could be observed by expanded imaging as two fluorescent regions separated by approximately 250 nm. This result indicated that the fluorescent signals beyond the optical limit were detected with higher resolution scanning by SNOM/AFM.

Chromosomes↗

Transfer-printing of highly aligned DNA nanowires.

We developed a simple method of reproducibly creating highly aligned DNA nanowires without any surface modifications or special equipment. Stretched DNA molecules initially present on the PDMS sheet were transferred onto another surface using transfer-printing (TP). Fluorescent microscopic and atomic force microscopic images revealed that many DNA molecules were highly aligned on surfaces after TP. Furthermore, it was also possible to realize the two-dimensional assembly of DNA nanowires by repeating TP.

Bacteriophage lambda↗

Hierarchical chromatin structure of Schizosaccharomyces pombe revealed by atomic force microscopy.

Many structural studies on higher eukaryotic chromatin have been carried out, but chromatin structure in fungi remains unclear. Schizosaccharomyces pombe has been used for investigations of chromosome function; however, the structural details of S. pombe chromatin have not been clarified owing to its small nucleus. We used atomic force microscopy for nano-scale imaging of chromatin isolated from S. pombe. Topographic images indicated that nuclear chromatin contained at least three hierarchical structures: large-scale chromatin fibers, spherical domains in the fibers, and nodules in the domains. The average diameters of the domain and the nodule were 363 +/- 85.2 nm and 46.2 +/- 9.30 nm. Each structure comprising the hierarchy was similar to higher eukaryotic chromatin thus far observed, despite definite differences in chromatin organization at the nucleosomal level. The presence of histone H1 suggested that there might be an alternative to compensate for histone H1 lacking in S. pombe.

Cell Cycle↗

Atomic force microscopy study of chromosome surface structure changed by protein extraction.

We applied atomic force microscopy (AFM) to investigate the surface structure of barley chromosome in combination with a chemical treatment method. As a result, we have obtained high-resolution topographic images of granular structures with a diameter of ca. 50 nm on the surface of critical-point dried metaphase chromosomes. Treatment with 2M NaCl significantly modified the chromosome surface structure: surface roughness was increased and chromosome thickness was decreased. The NaCl treatment extracted two major proteins with molecular weights of 4000 and 20,000 Da. These proteins might be belonging to non-histone protein families that do not contain any aromatic amino acid. The results demonstrate the advantage of the combined method of high-resolution AFM imaging and chemical treatments for understanding nano-scale surface structures of the chromosome.

Chromosome Structures↗

Analysis by atomic force microscopy of morphological changes in barley chromosomes during FISH treatment.

We employed atomic force microscopy (AFM) to examine structural changes in barley chromosomes during the four steps of standard FISH processes. Rehydration and dehydration with alcohol accompanying RNase treatment increased chromosome arm width and decreased chromosome height about 50%. Subsequent heat denaturation reduced chromosome height further. These three-dimensional structural changes of the chromosomes were substantial, but the FISH signal produced by the hybridization of fluorescent probes was clear when observed by a fluorescence microscope. In higher-magnification images, we observed granular structures considered to represent the chromatin fiber on the surface of the chromosomes in each FISH protocol step. These our results indicate that FISH treatments result in severe damage of the three-dimensional higher-order structures of the chromosomes, although nano-structures, such as nucleosome and chromatin fibers, remain intact and relatively unaffected.

Artifacts↗

Method for patterning stretched DNA molecules on mica surfaces by soft lithography.

Lambda DNA was stretched and patterned on mica surface using soft lithography. A highly diluted solution of amino propyl trimethoxy silane in hexane was deposited on a line patterned polydimethylsiloxane (PDMS) stamp. The functionalized stamp was then used to pick up DNA by molecular combing while the line patterns are parallel to the liquid surface. The stamp was then microcontact printed on freshly cleaved mica. We successfully obtained stretched DNA pattern on mica surface. DNA was found to be stretched in patterns perpendicular to those carved on the stamp. The stretched DNA population was large enough to be used for molecular biology mapping studies. Furthermore, the possibility of locating stretched DNA molecules in the desired position by stamping makes this method a good candidate for assembling non-semiconductor molecular devices.

Aluminum Silicates↗

P-cadherin expression in skin peeled with phenol or trichloroacetic acid (TCA).

P-cadherin expression patterns were studied in trichloroacetic acid (TCA) or phenol treated skin. The expression was absent or very weak on the basal cell surfaces by day 2. Seven days after peeling, P-cadherin was clearly distributed in a continuous granular pattern over the cell surface of the entire epidermis of the 40% TCA treated skin, in a weak granular pattern on a few suprabasal cells and basal cells of the phenol treated skin, and very weakly expressed on the lateral surfaces and in the cytoplasm of basal cells of the 60% TCA treated skin. Based on the present results and previous reports, it is likely that there are distinct patterns of P cadherin expression. Furthermore, a specific type of P cadherin expression might be involved in wound healing in general, which could provide new insights into tissue repair mechanisms after chemical peeling.

Adult↗

Unique therapeutic effects of the Japanese-Chinese herbal medicine, Sairei-to, on Th1/Th2 cytokines balance of the autoimmunity of MRL/lpr mice.

Sairei-to, one of the Japanese-Chinese herbal medicines has been used for the treatment of various diseases, especially collagen disease and edema in nephrotic syndrome. However, the mechanism of the therapeutic effects remains uncertain. Therefore, we investigated the immunological changes of skin, kidney, spleen cells and serum in autoimmune-prone MRL/lpr, MRL/n and C57BL/6J mice treated with Sairei-to. In MRL/lpr mice treated with Sairei-to, the improvement of proteinuria, reduction in the number of hematoxylin bodies in kidney, and reduced serum levels of blood urea nitrogen were observed. These results indicate that Sairei-to can improve or inhibit the progression of lupus nephritis. The proportion of CD19 and the serum levels of IgG1, which is one of the pathogenesis of lupus dermatoses and lupus nephritis, were significantly reduced in Sairei-to-treated MRL/lpr mice. Therefore, it is suspected that the B cell function was suppressed by Sairei-to. In addition, CD4/8 ratio in spleen cells and the degree of lymphoproliferation in MRL/lpr mice also decreased. Interestingly, IL-4 producing spleen cells were increased significantly by ELISPOT assay, and IFN-gamma mRNA expressions were reduced in Sairei-to-treated MRL/lpr mice. Regarding the Th balance, an imbalance towards Th1 predominance may play a significant role in MRL/lpr mice, and the Th1 axis was suppressed and the Th2 axis became predominant in Sairei-to-treated MRL/lpr mice. On the other hand, Th2 cell type immunoglobulins (IgG1) were suppressed. These results suggested that Sairei-to is potential for impairing shifted Th1/Th2 balance and hypergammaglobulinemia resulting in therapeutic effects.

Animals↗

Comparison of anti 60 and 52 kDa SS-A/Ro antibodies in the pathogenesis of cutaneous lupus erythematosus.

Anti SS-A/Ro antibodies are involved in the pathogenesis of cutaneous lupus erythematosus (CLE) in a part through antibody dependent cellular cytotoxicity (ADCC). However, it is still obscure which and how anti 60 and anti 52 kDa Ro antibodies are involved. To address the issue, we examined both types of anti Ro antibodies of sera from patients with systemic LE (SLE) or discoid LE (LE). The titer of anti 60 kDa antibody in SLE was significantly much higher than that of DLE or control. The positive ratio of DLE showed more higher tendency than control, but it was not statistically significant. The similar tendency was observed in the titer of anti 52 kDa antibody. An association between the anti 60 kDa antibody and the anti 52 kDa antibody was statistically significant in SLE patients. Although the relative index (RI) was statistically significant in DLE, they included many negative sera, which biased the statistics. Both anti 60 kDa antibody and anti 52 kDa antibody could induce significant ADCC of ultraviolet B (UVB) light-irradiated keratinocytes from neonatal foreskins and/or normal adults, in which anti 60 kDa antibody showed higher cytotoxicity than anti 52 kDa antibody. The autologous combination studies (keratinocytes and monospecific anti 60 or anti 52 kDa antibody from patients) suggested anti 60 kDa antibody was more potent to induce ADCC activity than anti 52 kDa antibody. Taken together, it is likely that anti 60 kDa antibody dependent keratinocyte damage plays more significant role in the pathogenesis of SLE skin lesions than anti 52 kDa dependent damage. However, both type antibodies seemed to have little contribution to the pathogenesis of DLE skin lesion.

Adult↗