Theory of robustness of irreversible differentiation in a stem cell system: chaos hypothesis

Based on an extensive study of a dynamical systems model of the development of a cell society, a novel theory for stem cell differentiation and its regulation is proposed as the "chaos hypothesis". Two fundamental features of stem cell systems-stochastic differentiation of stem cells and the robustness of a system due to regulation of this differentiation-are found to be general properties of a system of interacting cells exhibiting chaotic intra-cellular reaction dynamics and cell division, whose presence does not depend on the detail of the model. It is found that stem cells differentiate into other cell types stochastically due to a dynamical instability caused by cell-cell interactions, in a manner described by the Isologous Diversification theory. This developmental process is shown to be stable not only with respect to molecular fluctuations but also with respect to the removal of cells. With this developmental process, the irreversible loss of multipotency accompanying the change from a stem cell to a differentiated cell is shown to be characterized by a decrease in the chemical diversity in the cell and of the complexity of the cellular dynamics. The relationship between the division speed and the loss of multipotency is also discussed. Using our model, some predictions that can be tested experimentally are made for a stem cell system.     

G2 chromatid damage and repair kinetics in normal human fibroblast cells exposed to low- or high-LET radiation

Radiation-induced chromosome damage can be measured in interphase using the Premature Chromosome Condensation (PCC) technique. With the introduction of a new PCC technique using the potent phosphatase inhibitor calyculin-A, chromosomes can be condensed within five minutes, and it is now possible to examine the early damage induced by radiation. Using this method, it has been shown that high-LET radiation induces a higher frequency of chromatid breaks and a much higher frequency of isochromatid breaks than low-LET radiation. The kinetics of chromatid break rejoining consists of two exponential components representing a rapid and a slow time constant, which appears to be similar for low- and high-LET radiations. However, after high-LET radiation exposures, the rejoining process for isochromatid breaks influences the repair kinetics of chromatid-type breaks, and this plays an important role in the assessment of chromatid break rejoining in the G2 phase of the cell cycle.     

Automatic particle pickup method using a neural network has high accuracy by applying an initial weight derived from eigenimages: a new reference free method for single-particle analysis

The single-particle analysis is a structure-determining method for electron microscope (EM) images which does not require crystal. In this method, the projections are picked up and averaged by the images of similar Euler angles to improve the signal to noise ratio, and then create a 3-D reconstruction. The selection of a large number of particles from the cryo-EM micrographs is a pre-requisite for obtaining a high resolution. To pickup a low-contrast cryo-EM protein image, we have recently found that a three-layer pyramidal-type neural network is successful in detecting such a faint image, which had been difficult to detect by other methods. The connection weights between the input and hidden layers, which work as a matching filter, have revealed that they reflect characters of the particle projections in the training data. The images stored in terms of the connection weights were complex, more similar to the eigenimages which are created by the principal component analysis of the learning images rather than to the averages of the particle projections. When we set the initial learning weights according to the eigenimages in advance, the learning period was able to be shortened to less than half the time of the NN whose initial weights had been set randomly. Further, the pickup accuracy increased from 90 to 98%, and a combination of the matching filters were found to work as an integrated matching filter there. The integrated filters were amazingly similar to averaged projections and can be used directly as references for further two-dimensional averaging. Therefore, this research also presents a brand-new reference-free method for single-particle analysis.     

A sensitive non-radioactive in situ hybridization method for the detection of chicken IgG γ-chain mRNA: a technique suitable for detecting of variety of mRNAs in tissue sections

We established a sensitive non-radioactive in situ hybridization (ISH) method for the detection of chicken IgG γ-chain mRNA in paraffin sections. RNA probes were transcribed in vitro fromcloned chicken IgG CH1 nucleotide sequences with SP6/T7 RNA polymerases in the presence of DIG-UTP. These probes were used for hybridization and were immunodetected using anti-DIG antibodies conjugated to horseradish peroxidase. The immunoreactive products were visualized with DAB-H₂O₂. IgG γ-chain mRNA-expressing cells were localized in both the spleen and oviductal tissues. This method demonstrated an excellent sensitivity since the ISH signal was clear and the background was negligible. We found that in the spleen IgG γ-chain mRNA-expressing cells were present mainly in the red pulp, whereas in the oviduct they appeared mainly in the mucosal stroma and not in the mucosal epithelium. Published: May 14, 2001.     

Natural resistance to Clover yellow vein virus in beans controlled by a single recessive locus

We characterized the resistance of the common bean cv. Jolanda to Clover yellow vein virus no. 30 (ClYVV). After inoculation, the virus was detected in neither inoculated nor upper leaves, suggesting that the resistance operates at either the viral replication or cell-to-cell movement level. To analyze the mechanism of resistance, we developed a green fluorescent protein (GFP)-tagged ClYVV, and monitored GFP fluorescence at sites of infection on ClYVV-inoculated leaves. No GFP fluorescence was detected in Jolanda, whereas its expression in single cells and spread on inoculated leaves were observed clearly in susceptible cultivars. ClYVV-introduced Jolanda cells were found to be still viable; therefore, it is unlikely that the restriction of multiplication was due to rapid cell death. Genetic analysis indicated that a single recessive locus controlled the resistant phenotype of Jolanda. We designated this locus desc (determinant of susceptibility to ClYVV). Meanwhile, a spontaneous mutant virus that overcomes the resistance (ClYVV-Br) was isolated. Inoculation assays using chimeric viruses suggested that a viral genome-linked protein (VPg) might be the avirulence determinant. The resistance mechanism may be associated with the role of VPg in the viral infection cycle.     

Detection of 1270 nm emission from singlet oxygen and photocytotoxic property of sugar-pendant 60 fullerenes

Sugar-pendant [60] fullerene derivatives have been prepared from carbohydrate-linked azides 1a-e. Both monosugar (4a-e) and bissugar derivatives (5a-e) produce singlet oxygen ((1)O(2)) under laser irradiation (355 nm) proved by the direct observation of (1)O(2) emission at 1270 nm. Monosugar derivatives exhibit photocytotoxicity varying by the attached sugar molecule.     

Stilbenoids from the stem of Gnetum latifolium (Gnetaceae)

An acetone extract of the stem of Gnetum latifolium Blume afforded the stilbene trimer (latifolol) together with five known stilbenoids (gnetin E, gnetin D, gnetin C, (-)epsilon -viniferin and resveratrol). Their structures were elucidated on the basis of spectral evidence, in particular by using 2D NMR methods.     

Identification of target genes conferring ethanol stress tolerance to Saccharomyces cerevisiae based on DNA microarray data analysis

During industrial production process using yeast, cells are exposed to the stress due to the accumulation of ethanol, which affects the cell growth activity and productivity of target products, thus, the ethanol stress-tolerant yeast strains are highly desired. To identify the target gene(s) for constructing ethanol stress tolerant yeast strains, we obtained the gene expression profiles of two strains of Saccharomyces cerevisiae, namely, a laboratory strain and a strain used for brewing Japanese rice wine (sake), in the presence of 5% (v/v) ethanol, using DNA microarray. For the selection of target genes for breeding ethanol stress tolerant strains, clustering of DNA microarray data was performed. For further selection, the ethanol sensitivity of the knockout mutants in each of which the gene selected by DNA microarray analysis is deleted, was also investigated. The integration of the DNA microarray data and the ethanol sensitivity data of knockout strains suggests that the enhancement of expression of genes related to tryptophan biosynthesis might confer the ethanol stress tolerance to yeast cells. Indeed, the strains overexpressing tryptophan biosynthesis genes showed a stress tolerance to 5% ethanol. Moreover, the addition of tryptophan to the culture medium and overexpression of tryptophan permease gene conferred ethanol stress tolerance to yeast cells. These results indicate that overexpression of the genes for trypophan biosynthesis increases the ethanol stress tolerance. Tryptophan supplementation to culture and overexpression of the tryptophan permease gene are also effective for the increase in ethanol stress tolerance. Our methodology for the selection of target genes for constructing ethanol stress tolerant strains, based on the data of DNA microarray analysis and phenotypes of knockout mutants, was validated.