Kenichi Ijiri

MALAT‐1 enhances cell motility of lung adenocarcinoma cells by influencing the expression of motility‐related genes

MALAT‐1, a long non‐coding RNA, is associated with metastasis, but its role in the metastatic process remains unknown. Here, we show that short‐interfering RNA‐mediated MALAT‐1 silencing impaired in vitro cell motility of lung cancer cells and influenced the expression of numerous genes. In these genes, knockdown of any one of CTHRC1, CCT4, HMMR, or ROD1 clearly inhibited cell migration. In MALAT‐1 knockdown cells, pre‐mRNA levels were decreased in some but not all genes. Thus, our findings suggest that MALAT‐1 is a novel class of non‐coding RNA that promotes cell motility through transcriptional and post‐transcriptional regulation of motility related gene expression.

Identification of cis- and trans-acting factors involved in the localization of MALAT-1 noncoding RNA to nuclear speckles

MALAT-1 noncoding RNA is localized to nuclear speckles despite its mRNA-like characteristics. Here, we report the identification of several key factors that promote the localization of MALAT-1 to nuclear speckles and also provide evidence that MALAT-1 is involved in the regulation of gene expression. Heterokaryon assays revealed that MALAT-1 does not shuttle between the nucleus and cytoplasm. RNAi-mediated repression of the nuclear speckle proteins, RNPS1, SRm160, or IBP160, which are well-known mRNA processing factors, resulted in the diffusion of MALAT-1 to the nucleoplasm. We demonstrated that MALAT-1 contains two distinct elements directing transcripts to nuclear speckles, which were also capable of binding to RNPS1 in vitro. Depletion of MALAT-1 represses the expression of several genes. Taken together, our results suggest that RNPS1, SRm160, and IBP160 contribute to the localization of MALAT-1 to nuclear speckles, where MALAT-1 could be involved in regulating gene expression.

Identification of hundreds of novel UPF1 target transcripts by direct determination of whole transcriptome stability

UPF1 eliminates aberrant mRNAs harboring premature termination codons, and regulates the steady-state levels of normal physiological mRNAs. Although genome-wide studies of UPF1 targets performed, previous studies did not distinguish indirect UPF1 targets because they could not determine UPF1-dependent altered RNA stabilities. Here, we measured the decay rates of the whole transcriptome in UPF1-depleted HeLa cells using BRIC-seq, an inhibitor-free method for directly measuring RNA stability. We determined the half-lives and expression levels of 9,229 transcripts. An amount of 785 transcripts were stabilized in UPF1-depleted cells. Among these, the expression levels of 76 transcripts were increased, but those of the other 709 transcripts were not altered. RNA immunoprecipitation showed UPF1 bound to the stabilized transcripts, suggesting that UPF1 directly degrades the 709 transcripts. Many UPF1 targets in this study were newly identified. This study clearly demonstrates that direct determination of RNA stability is a powerful approach for identifying targets of RNA degradation factors.

The Circadian Rhythm for the Number and Sensitivity of Radiation-induced Apoptosis in the Crypts of Mouse Small Intestine

Apoptosis is a mode of cell death involving nuclear pycnosis, cytoplasmic condensation and karyorrhexis. Changes in the number of apoptotic cells at various times (3-12 h) after a single dose of either 0.5 or 9.0 Gy given at 09.00, 21.00 or 03.00 h were studied in histological sections of small intestinal crypts of mice. The incidences of apoptosis were examined 3 or 6 h after irradiation at different times of day with different doses of gamma-rays ranging from 0.15 to 9.0 Gy. Survival curves were constructed from the dose-incidence curves for apoptosis, using the number of apoptotic cells after high doses (NM) as the maximum cell population size. The mean lethal doses (Do) for the dose range 0-0.5 Gy were calculated for each time of day. A circadian rhythm in both Do and NM values was detected, indicating that both the number and sensitivity of radiation-induced apoptosis were changing throughout the day. A possible explanation based on the cell-cycle states of the target cell population for apoptosis (presumably functional stem cells) was drawn. Most of the target cells were assumed to be in an extended G1 phase. Around 21.00 h a transition from G1 to S phase takes place in some of these cells (approximately seven or eight cells per whole crypt). The S phase then lasts till around 06.00 h. They may be at G2 and M around 06.00-09.00 h, and then they re-enter G1. The circadian rhythm for the number and sensitivity of the cells susceptible to apoptosis obtained in the present report agrees well with this pattern of cell-cycle phases of target cells.

Effects of Irradiation on Germ Cells and Embryonic Development in Teleosts

Publisher Summary This chapter discusses the effects of irradiation on germ cells and embryonic development in teleosts. Sterility and weight reduction of testes after irradiation are mainly because of actual cell loss, and the order of decreasing sensitivity of spermatogenic cells is from the most sensitive spermatogonia, through primary and secondary spermatocytes and spermatids, to sperm, which are quite radioresistant. Even in morphologically homogeneous populations of spermatogonia, heterogeneity of sensitivity exists. The results of local irradiation of the testes are different from those of irradiation of the ovaries. The radiosensitivity of gametogenesis in the ovary and testis cannot be directly compared, except probably from the viewpoint of genetic effects of radiation. Most studies concerning radiosensitivity of fish embryos have so far been performed for the end points of hatchability and lethality during development. This is mainly because of the simplicity of the technique. Many irradiated larvae that appear normal at hatching carry radiation damage that becomes evident at some future stage.

Identification and Characterization of Novel Genotoxic Stress-Inducible Nuclear Long Noncoding RNAs in Mammalian Cells

Whole transcriptome analyses have revealed a large number of novel transcripts including long and short noncoding RNAs (ncRNAs). Currently, there is great interest in characterizing the functions of the different classes of ncRNAs and their relevance to cellular processes. In particular, nuclear long ncRNAs may be involved in controlling various aspects of biological regulation, such as stress responses. By a combination of bioinformatic and experimental approaches, we identified 25 novel nuclear long ncRNAs from 6,088,565 full-length human cDNA sequences. Some nuclear long ncRNAs were conserved among vertebrates, whereas others were found only among primates. Expression profiling of the nuclear long ncRNAs in human tissues revealed that most were expressed ubiquitously. A subset of the identified nuclear long ncRNAs was induced by the genotoxic agents mitomycin C or doxorubicin, in HeLa Tet-off cells. There were no commonly altered nuclear long ncRNAs between mitomycin C- and doxorubicin-treated cells. These results suggest that distinct sets of nuclear long ncRNAs play roles in cellular defense mechanisms against specific genotoxic agents, and that particular long ncRNAs have the potential to be surrogate indicators of a specific cell stress.

Circadian Rhythms in the Incidence of Apoptotic Cells and Number of Clonogenic Cells in Intestinal Crypts after Radiation Using Normal and Reversed Light Conditions

Variations in the number of radiation-induced morphologically dead or dying cells (apoptotic cells) in the crypts in the small intestine of the mouse have been studied throughout a 24-h period under a normal light regimen (light on, 07.00-19.00 h; light off, 19.00-07.00 h). A clear circadian rhythm was displayed in the apoptotic incidence 3 or 6 h after irradiation for each gamma-ray dose studied (range 0.14-9.0 Gy). The most prominent circadian rhythm was obtained after 0.5 Gy. The peak time of day for inducing apoptosis was 06.00-09.00 h, and the trough occurred at 18.00-21.00 h. Some mice were also transferred to a room with the light cycle reversed, and were irradiated on different days after the transfer. The apoptosis induced by 0.5 Gy or 9.0 Gy, or the number of surviving crypts (microcolonies) after 11.0 Gy or 13.0 Gy was examined. The transition point for reversal (i.e. the switch time from the normal-light pattern to the reversed-light pattern) of the circadian rhythm in apoptosis (after 0.5 Gy) occurred 7 days after the transfer and the rhythm was reversed by 14 days. The rhythm for crypt survival (i.e. for clonogenic cell radiosensitivity) was disturbed on 1 day and the transition point for reversal occurred 3 days after the transfer. The rhythm became reversed by 7 days. These observations are discussed in relation to the identity of clonogenic cells, (functional) stem cells, proliferating transit cells and the cells sensitive to small doses of radiation (i.e. hypersensitive cells) in the crypt.

Aquatic animal research in space station and its issues — focus on support technology on nitrate toxicity —

We studied the effects of accumulated nitrate in water on the spawning, hatching and development of medaka using a simple nitrifying filter and a combined filter having both nitrifying and denitrifying capabilities. A nitrate concentration of 100 mg NO3(-)-N/L was clearly of lethal toxicity to fish when they were exposed to nitrate in both adult and the growing phases. A nitrate concentration of 75 mg NO3(-)-N/L reduced the fertilization rate, delayed the hatching time and reduced the hatching rate of the eggs laid by adults and decreased the growth rate of juveniles. In addition, nitrate accumulations as low as 50 mg NO3(-)-N/L remarkably retarded spawning and lowered the number of eggs laid by fish exposed in the juvenile phase. The effects on the reproduction system may be initiated by a low concentration, approximately 30 mg NO3(-)-N/L.

The re-establishment of hypersensitive cells in the crypts of irradiated mouse intestine

Within 3-6 h of small doses of radiation (gamma-rays) the number of dead cells (apoptotic cells) in the crypts of the small intestine reaches peak values. These return to normal levels only after times later than 1 day. After higher doses elevated levels of cell death persist for longer times. The dead cells first occur most frequently at the lower positions of the crypt (median value for the distribution of apoptotic fragments is about cell position 6). At later times more dead cells are observed at higher positions. Two doses of radiation separated by various time intervals have been used to investigate when after irradiation the cell population susceptible to acute cell death is re-established. Dead cells were scored 3 or 6 h after the second dose. The yield of dead cells after two doses represents the sum of the dead cells produced by, and persisting from, the first dose and new apoptotic cells induced by the second dose. Since the temporal and dose-dependence aspects of the dead-cell yield after the first dose alone is known, the additional dead cells attributable to the second dose alone can be determined by subtraction. Within 1-2 days of small doses (0.5 Gy) the sensitive cells, recognized histologically as apoptotic cells, are re-established at the base of the crypt (around cell position 6). After higher doses (9.0 Gy) they are not re-established until about the fourth day after irradiation. Even in the enlarged regenerating crypts the sensitive cells are found at the same position at the crypt base. It has been estimated that the crypt contains five or six cells that are susceptible to low doses (0.5 Gy) (hypersensitive cells) and up to a total of only seven or eight susceptible cells that can be induced by any dose to enter the sequence of changes implicit in apoptosis. Between 4 and 10 days after an initial irradiation of 9.0 Gy the total number of susceptible cells increased from seven to eight to about 10 to 13 per crypt.

Vestibular and visual contribution to fish behavior under microgravity.

Vestibular and visual information are two major factors fish use for controlling their posture under 1 G conditions. Parabolic flight experiments were carried out to observe the fish behavior under microgravity for several different strains of Medaka fish (Oryzias latipes). There existed a clear strain-difference in the behavioral response of the fish under microgravity: Some strains looped, while other strains did not loop at all. However, even the latter strains looped under microgravity conditions when kept in complete darkness, suggesting the contribution of visual information to the posture control under microgravity. In the laboratory, eyesight (visual acuity) was checked for each strain, using a rotating striped-drum apparatus. The results also showed a strain-difference, which gave a clue to the different degree of adaptability to microgravity among different strains. Beside loopings, some fish exhibited rolling movement around their body axis. Tracing each fish during and between parabolas, it was shown that to which side each fish rolls was determined specifically to each individual fish, and not to each strain. Thus, rolling direction is not genetically determined. This may support the otolith asymmetry hypothesis. Fish of a mutant strain (ha strain, having homozygous recessive of one gene ha) have some malfunction in otolith-vestibular system, and their behavior showed they are not dependent on gravity. Morphological abnormalities of their ear vesicles during the embryonic and baby stages were noted. Their eyesight and dorsal light responses were also studied. Progress in the project of establishing a new strain which has good eyesight and, at the same time, being deficient in otolith-vestibular system was reported. Crosses between the strain of good eyesight and ha strain were made, and to some extent, F2 fish have already shown such characteristics suited for living under microgravity conditions.