Visualisation of cell cycle modifications by X-ray irradiation of single HeLa cells using fluorescent ubiquitination-based cell cycle indicators.

To explore the effects of X-ray irradiation on mammalian cell cycle dynamics, single cells using the fluorescent ubiquitination-based cell cycle indicator (Fucci) technique were tracked. HeLa cells expressing Fucci were used to visualise cell cycle modifications induced by irradiation. After cultured HeLa-Fucci cells were exposed to 5 Gy X-rays, fluorescent cell images were captured every 20 min for 48 h using a fluorescent microscope. Time dependence of the fluorescence intensity of S/G2 cells was analysed to examine the cell cycle dynamics of irradiated and non-irradiated control cells. The results showed that irradiated cells could be divided into two populations: one with similar cell cycle dynamics to that of non-irradiated cells, and another displaying a prolonged G2 phase. Based on these findings, it is proposed in this article that an underlying switch mechanism is involved in cell cycle regulation and the G2/M checkpoint of HeLa cells.

Live-cell imaging study of mitochondrial morphology in mammalian cells exposed to X-rays.

Morphological changes in mitochondria induced by X-irradiation in normal murine mammary gland cells were studied with a live-cell microscopic imaging technique. Mitochondria were visualised by staining with a specific fluorescent probe in the cells, which express fluorescent ubiquitination-based cell-cycle indicator 2 (Fucci2) probes to visualise cell cycle. In unirradiated cells, the number of cells with fragmented mitochondria was about 20 % of the total cells through observation period (96 h). In irradiated cells, the population with fragmented mitochondria significantly increased depending on the absorbed dose. Particularly, for 8 Gy irradiation, the accumulation of fragmentation persists even in the cells whose cell cycle came to a stand (80 % in G1 (G0-like) phase). The fraction reached to a maximum at 96 h after irradiation. The kinetics of the fraction with fragmented mitochondria was similar to that for cells in S/G2/M phase (20 %) through the observation period (120 h). The evidences show that, in irradiated cells, some signals are continually released from a nucleus or cytoplasm even in the G0-like cells to operate some sort of protein machineries involved in mitochondrial fission. It is inferred that this delayed mitochondrial fragmentation is strongly related to their dysfunction, and hence might modulate radiobiological effects such as mutation or cell death.

Temporal and spatial localization of novel nuclear protein NP95 in mitotic and meiotic cells.

Expression of novel NP95 (nuclear protein, 95 kDa), which contains a leucine zipper motif, a zinc finger motif, a putative cyclin A/E-cyclin-dependent protein kinase 2 phosphorylation site, and retinoblastoma protein-binding motifs, is associated with S-phase progression of mouse cells. It is suppressed during G1 and G2/M phases in normal thymocytes but expressed at a constantly high level irrespective of cell stage in mouse T cell lymphoma cells. NP95 was shown previously to be expressed strongly only in proliferative tissues and cells. In this immunohistochemical study, we demonstrate that NP95 is localized in S-phase nuclei as dot-like foci. Double immunostaining of NP95 and proliferating cell nuclear antigen (PCNA) showed that NP95 was co-localized with PCNA. Construction of three-dimensional images indicated that NP95 was localized with PCNA in replication sites in a somewhat distinct temporal manner. During meiosis, NP95 was present not only in proliferating spermatogonia but also in meiotic spermatocytes and differentiating spermatids which were not proliferating. The possible role of NP95 in mitotic and meiotic cells is discussed.

Process development for industrial-scale preparation of carboxypeptidase Y secreted by Saccharomyces cerevisiae.

A method for industrial-scale preparation of carboxypeptidase Y (CPY), which was secreted by Saccharomyces cerevisiae KS58-2D/pCY303 into the culture broth, was developed. Because the purification process consists of a few simple unit operations including only one chromatography step, a higher CPY recovery was achieved than that in the process using disrupted baker's yeast. Approximately 100 g of purified CPY powder was constantly obtained using the final culture broth from a 500-l fermentor.

Variable gene segment-specific N-insertions at the signal joint of T-cell receptor Vbeta-Dbeta recombinations.

The V(D)J recombination of immunoglobulin and a T-cell receptor generates two species of DNA junctions, a coding joint and a signal joint. Non-templated nucleotides (N-nucleotides) are inserted in these DNA junctions. We analyzed the N-insertion at signal joints generated by the Vbeta-Dbeta recombinations. N-insertions were detected at signal joints of Vbeta2, Vbeta3, Vbeta10, Vbeta18 and Vbeta14 but not in Vbeta8 and Vbeta7. These data show that the N-insertion at signal joints is dependent on the Vbeta locus used for the recombination. We suggest that the regional chromosomal configuration may differ in recombinase accessibility.

Transforming growth factor-beta induced class switch recombination may be modified during the cell proliferation restored by IL-2 stimulation.

Mouse spleen cells were stimulated, first by transforming growth factor beta (TGF-beta) and then by interleukin-2 (IL-2) in the presence of bacterial lipopolysaccharide (LPS). LPS plus IL-2 stimulation restored the cell proliferation suppressed by TGF-beta and increased the number of B cells. Both TGF-beta alone or TGF-beta plus IL-2 enhanced production of surface IgA-positive (sIgA+) cells in LPS-stimulated B-cell cultures. We characterized extrachromosomal circular DNAs generated in TGF-beta-primed and IL-2-stimulated B cells and identified the breakpoints of S mu/S gamma 3 recombinants which were the major intermediates of S mu and S alpha switch recombination. All switch recombination sites were dispersed evenly within both S mu and S gamma 3 regions. This even distribution of S mu/S gamma 3 breakpoints is in contrast to the site preference of class switch breakpoints induced by TGF-beta alone. These results suggest that IL-2-stimulated cell proliferation may modify class switch recombination primed by TGF-beta.

Molecular characterization of extrachromosomal circular DNAs from an embryonal carcinoma cell line induced to differentiate into neuron-like cells in vitro.

Extrachromosomal circular DNAs isolated from a P19 embryonal carcinoma cell line were induced to differentiate into neuron-like cells by retinoic acid and cloned into an EcoRI site of a phage vector. Of the 26 DNA inserts (2.1 kb in average length) analyzed, 16 contained repetitive sequences. Out of 10 DNA inserts with unique sequence, 6 carried linear chromosomal sequences and 4 showed chromosomal rearrangements in Southern blots. Two unique fragments with germline configuration were enriched in circular DNA clone libraries. We assigned the breakpoints of 3 circular DNA fragments to positions in the germline sequence. Patchy short inverted repeats were found in the vicinity of breakpoints. An intrastrand loop structure between such inverted short homology region may be required for the circularization of excised DNA.