Early development and neurogenesis of Temnopleurus reevesii

Sea urchins are model non‐chordate deuterostomes, and studying the nervous system of their embryos can aid in the understanding of the universal mechanisms of neurogenesis. However, despite the long history of sea urchin embryology research, the molecular mechanisms of their neurogenesis have not been well investigated, in part because neurons appear relatively late during embryogenesis. In this study, we used the species Temnopleurus reevesii as a new sea urchin model and investigated the detail of its development and neurogenesis during early embryogenesis. We found that the embryos of T. reevesii were tolerant of high temperatures and could be cultured successfully at 15–30°C during early embryogenesis. At 30°C, the embryos developed rapidly enough that the neurons appeared at just after 24 h. This is faster than the development of other model urchins, such as Hemicentrotus pulcherrimus or Strongylocentrotus purpuratus. In addition, the body of the embryo was highly transparent, allowing the details of the neural network to be easily captured by ordinary epifluorescent and confocal microscopy without any additional treatments. Because of its rapid development and high transparency during embryogenesis, T. reevesii may be a suitable sea urchin model for studying neurogenesis. Moreover, the males and females are easily distinguishable, and the style of early cleavages is intriguingly unusual, suggesting that this sea urchin might be a good candidate for addressing not only neurology but also cell and developmental biology.     

Proliferative kinetics and differentiation of murine blast cell colonies in culture: Evidence for variable G0 periods and constant doubling rates of early pluripotent hemopoietic progenitors

Several investigators have described hemopoietic colonies expressing multilineage differentiation in culture. We recently identified a class of murine hemopoletic progenitors which form blast cell colonies with very high replating efficiencies. In order to clarify further the relationship between progenitors for blast cell colonies and progenitors for the multilineage hemopoietic colonies in culture, we carried out analyses of kinetic and differentiation properties of murine blast cell colonies. Serial observations of the development of blast cell colonies into multilineage (and single lineage) colonies in cultures of spleen cells obtained from 5-fluorouracil (5-FU)-treated mice confirmed the transitional nature of the murine blast cell colonies. The data also suggested that the early pluripotent progenitors are in G₀ for variable periods, and that when triggered into cell cycle, they proliferate at relatively constant doubling rates during the early stages of differentiation. The notion that some of the pluripotent progenitors are in G₀ was also supported by long-term thymidine suicide studies in which spleen cells were exposed to ³H-thymidine with high specific activity for 5 days in culture, washed, and assayed for surviving progenitors. Comparison of replating abilities of day-7 and day-16 blast cell colonies from normal as well as 5-FU-treated mice indicated that some of the day-7 blast cell colonies are derived from maturer populations of progenitors which are sensitive to 5-FU. In contrast, progenitors for the day-16 blast cell colonies are dormant in cell cycle and were not affected by 5-FU treatment. Previously we reported that progenitors for day-16 blast cell colonies have a significant capacity for self-renewal. These observations suggest the hypothesis that the capability for self-renewal is accompanied by long periods of G₀, and that once commitment to differentiation takes place, then active cell division occurs.     

A temperature-sensitive mutant of the mammalian RNA helicase,DEAD-BOX X isoform,DBX, defective in the transition from G1 to S phase

ts ET24 cells are a novel temperature-sensitive (ts) mutant for cell proliferation of hamster BHK21 cells. The human genomic DNA which rescued the temperature-sensitive lethality of ts ET24 cells was isolated and screened for an open reading frame in the deposited human genomic library. X chromosomal DBX gene encoding the RNA helicase, DEAD-BOX X isoform, which is homologous to yeast Ded1p, was found to be defective in this mutant. The single point mutation (P267S) was localized between the Motifs I and Ia of the hamster DBX of ts ET24 cells. At the nonpermissive temperature of 39.5 degrees C, ts ET24 cells were arrested in the G1-phase and survived for more than 3 days. In ts ET24 cells, total protein synthesis was not reduced at 39.5 degrees C for 24 h, while mRNA accumulated in the nucleus after incubation at 39.5 degrees C for 17 h. The amount of cyclin A mRNA decreased in ts ET24 cells within 4 h after the temperature shift to 39.5 degrees C, consistent with the fact that the entry into the S-phase was delayed by the temperature shift.     

An In Vitro ES Cell-Based Clock Recapitulation Assay Model Identifies CK2α as an Endogenous Clock Regulator

We previously reported emergence and disappearance of circadian molecular oscillations during differentiation of mouse embryonic stem (ES) cells and reprogramming of differentiated cells, respectively. Here we present a robust and stringent in vitro circadian clock formation assay that recapitulates in vivo circadian phenotypes. This assay system first confirmed that a mutant ES cell line lacking Casein Kinase I delta (CKIδ) induced ∼3 hours longer period-length of circadian rhythm than the wild type, which was compatible with recently reported results using CKIδ null mice. In addition, this assay system also revealed that a Casein Kinase 2 alpha subunit (CK2α) homozygous mutant ES cell line developed significantly longer (about 2.5 hours) periods of circadian clock oscillations after in vitro or in vivo differentiation. Moreover, revertant ES cell lines in which mutagenic vector sequences were deleted showed nearly wild type periods after differentiation, indicating that the abnormal circadian period of the mutant ES cell line originated from the mutation in the CK2α gene. Since CK2α deficient mice are embryonic lethal, this in vitro assay system represents the genetic evidence showing an essential role of CK2α in the mammalian circadian clock. This assay was successfully applied for the phenotype analysis of homozygous mutant ES cells, demonstrating that an ES cell-based in vitro assay is available for circadian genetic screening.     

Prevention of PERV infections in pig to human xenotransplantation by the RNA interference silences gene

The possibility of preventing the transmission of porcine endogenous retrovirus (PERV) to human cells using short interfering RNAs (siRNA) was investigated. The siRNA for the p30 of PERV gag region was cloned into pSUPER, the polymerase-III H1-RNA gene promoter. A green fluorescence protein (GFP) was also cloned into pSUPER to establish pSXGH. Pig endothelial cells (PEC) were transduced with the LacZ gene by pseudotype infection, and infected with PERV subtype B, resulting in the formation of PEC(LacZ)/PB. The PEC(LacZ)/PB was next transfected with pSXGH-siRNA. The expression of siRNA was provisionally checked by determining the level of expression of GFP. Culture supernatants of infected cells were then inoculated into HEK293 cells. The siRNA clearly destroyed the PERV infectivity of PEC(LacZ)/PB in both transient cell lines and stable clones. Moreover, the decreased levels of mRNA and gag protein were evidenced in the stable clones by real-time PCR and Western blotting, respectively. The final goal of our study was to establish a transgenic pig expressing the siRNA for PERV. The results suggest that siRNA represents a novel approach for controlling PERV infections in clinical xenotransplantation.     

Identification and mapping of functional domains on human T-cell lymphotropic virus type 1 envelope proteins by using synthetic peptides.

To identify the regions that are important in human T-cell leukemia virus type 1 (HTLV-1) envelope function, we synthesized 23 kinds of peptides covering the envelope proteins and examined the inhibitory effect of each peptide on syncytium formation induced by HTLV-1-bearing cells. Of the 23 synthetic peptides, 2, corresponding to amino acids 197 to 216 on gp46 and 400 to 429 on gp21, inhibited syncytium formation induced by HTLV-1-bearing cells but did not affect syncytium formation induced by human immunodeficiency virus type 1-producing cells. The peptide concentrations giving 50% inhibition of syncytium formation for gp46 197 to 216 and gp21 400 to 429 were 14.9 and 6.0 microM, respectively. A syncytium formation assay with overlapping synthetic peptides containing amino acids 175 to 236 and 391 to 448 of the envelope proteins showed that syncytium formation was inhibited by peptides that contained the amino acid sequences 197 to 205 (Asp-His-Ile-Leu-Glu-Pro-Ser-Ile-Pro) and 397 to 406 (Gln-Glu-Gln-Cys-Arg-Phe- Pro-Asn-Ile-Thr). These observations suggest that the two regions corresponding to amino acids 197 to 216 and 400 to 429 are involved] in HTLV-1 envelope function.     

TPRA40/GPR175 regulates early mouse embryogenesis through functional membrane transport by Sjögren's syndrome-associated protein NA14

TPRA40/GPR175 is an orphan receptor whose physiological functions have not been found to date. In an attempt to generate transgenic mice that express an shRNA of TPRA40, we observed that the cell division of early mouse embryos that injected the short hairpin RNA expression vector was significantly accelerated compared with the control vector. The regulation of cell division by TPRA40 was also observed in HeLa cells. Since the C-terminal region of TPRA40 has been shown to be essential for the regulation of cell division, we performed yeast two-hybrid screening using the C-terminal region as bait. Nuclear antigen of 14 kDa (NA14), an autoantigen of Sj?gren's syndrome, was identified as a binding protein to the C-terminal region of TPRA40. The binding of TPRA40 and NA14 was confirmed by GST pull-down assay and co-immunoprecipitation assay. FLAG-TPRA40 is transported from the cytosol to the plasma membrane in time-dependent manner and the translocation was inhibited by GFP-NA14DeltaN, an N-terminal deletion mutant that cannot bind to microtubules but binds to TPRA40. TPRA40DeltaC, which cannot bind to NA 14, shows impaired transport to the plasma membrane. Finally, we found that the effect of TPRA40 on mouse embryogenesis is strengthened by GFP-NA14, but not by GFP or GFP-NA14DeltaN. These observations indicate that the functional plasma membrane transport of TPRA40 that regulates cell division of mouse embryos is mediated by NA14.     

Genetic Variation in VP7 Gene of Human Rotavirus Serotype 2 (G2 Type) Isolated in Japan,China, and Pakistan

Sequence analysis of the gene encoding the major neutralization glycoprotein (VP7) was performed on sixteen human isolates of serotype 2 of rotavirus in Japan, China, and Pakistan and their genetic variations were examined. Comparative studies of their nucleotide and deduced amino acid sequences between the sixteen isolates and the HU5 strain revealed an overall homology of more than 94%. A higher degree of homology in nucleotides was observed among the sixteen isolates than between HU5 and the isolates. A total of thirteen amino acid residues frequently converted to another amino acid. Out of the thirteen, five amino acid residues belonging to the major neutralizing epitope regions (C, E, and F in this communication) converted frequently. From the amino acid sequences three subtypes, subtype 1, subtype 2, and intermediate, were suggested to be classified as previously reported for serotype 1 (Xin et al, Virology, 1993, 197: 813-816).