DDX1 vesicles control calcium-dependent mitochondrial activity in mouse embryos.

The DEAD box protein DDX1, previously associated with 3'-end RNA processing and DNA repair, forms large aggregates in the cytoplasm of early mouse embryos. Ddx1 knockout causes stalling of embryos at the 2-4 cell stages. Here, we identify a DDX1-containing membrane-bound calcium-containing organelle with a nucleic acid core.

Nuclear-to-cytoplasmic ratios of 1PN and 2PN zygotes after fertilization of mouse oocytes.

Numerous studies have reported comparisons of the nuclear-to-cytoplasmic (NC) ratio during mitosis. However, little information is known about how the pronuclear size is regulated and determined at the end of meiosis II in mammalian zygotes. The present study aims to analyze the NC ratio of female and male pronuclei, and also to compare the size of single pronuclei using photographs that were obtained during experiments to create chimeric hermaphrodites from 2-cell oocytes.

Cytoplasmic aggregation of DDX1 in developing embryos: Early embryonic lethality associated with Ddx1 knockout.

Temporally-regulated maternal RNA translation is essential for embryonic development, with defective degradation resulting in stalled 2-cell embryos. We show that DDX1, a DEAD box protein implicated in RNA transport, may be a key regulator of maternal RNA utilization. DDX1 protein localizes exclusively to cytoplasmic granules in both oocytes and early stage mouse embryos, with DDX1 requiring RNA for retention at these sites.

Effective use of the TSPY gene-specific copy number in determining fetal DNA in the maternal blood of cynomolgus monkeys.

Since the available concentration of single-copy fetal genes in maternal blood DNA is sometimes lower than detection limits by PCR methods, the development of specific and quantitative PCR detection methods for fetal DNA in maternal blood is anticipated, which may broaden the methods that can be used to monitor pregnancy. We used the TaqMan qPCR amplification for DYS14 multi-copy sequence and the SRY gene in maternal blood plasma (cell-free DNA) and fractional precipitated blood cells (cellular DNA) from individual cynomolgus monkeys at 22 weeks of pregnancy. The availability of cell-free fetal DNA was higher in maternal blood plasma than that of cellular DNA from fractional precipitated blood cells.

Detection and quantification of male-specific fetal DNA in the serum of pregnant cynomolgus monkeys (Macaca fascicularis).

Because of their developmental similarities to humans, nonhuman primates are often used as a model to study fetal development for potential clinical applications in humans. The detection of fetal DNA in maternal plasma or serum offers a source of fetal genetic material for prenatal diagnosis. However, no such data have been reported for cynomolgus monkeys (Macaca fascicularis), an important model in biomedical research.

Naïve-like conversion enhances the difference in innate in vitro differentiation capacity between rabbit ES cells and iPS cells.

Quality evaluation of pluripotent stem cells using appropriate animal models needs to be improved for human regenerative medicine. Previously, we demonstrated that although the in vitro neural differentiating capacity of rabbit induced pluripotent stem cells (iPSCs) can be mitigated by improving their baseline level of pluripotency, i.e.

Single-step generation of rabbits carrying a targeted allele of the tyrosinase gene using CRISPR/Cas9.

Targeted genome editing of nonrodent mammalian species has provided the potential for highly accurate interventions into gene function in humans and the generation of useful animal models of human diseases. Here we show successful clustered regularly interspaced short palindromic repeat (CRISPR) and CRISPR-associated (Cas)-mediated gene targeting via circular plasmid injection in rabbits. The rabbit tyrosinase gene (TYR) was effectively disrupted, and we confirmed germline transmission by pronuclear injection of a circular plasmid expressing humanized Cas9 (hCas9) and single-guide RNA.

Role of retinoic acid and fibroblast growth factor 2 in neural differentiation from cynomolgus monkey (Macaca fascicularis) embryonic stem cells.

Retinoic acid is a widely used factor in both mouse and human embryonic stem cells. It suppresses differentiation to mesoderm and enhances differentiation to ectoderm. Fibroblast growth factor 2 (FGF2) is widely used to induce differentiation to neurons in mice, yet in primates, including humans, it maintains embryonic stem cells in the undifferentiated state.

Symmetrical division of mouse oocytes during meiotic maturation can lead to the development of twin embryos that amalgamate to form a chimeric hermaphrodite.

Background: Gentle compression of mouse oocytes during meiosis-1 prevented the usual extrusion of a small polar body and resulted in the symmetrical division of the ooplasm into two cells of similar size within the zona pellucida. The purpose of our study was to determine whether such cells, equivalent to two small oocytes, were capable of embryonic development and would result in birth following transfer to the uterus.

Methods: IVF of the 2-celled oocytes was performed and the twin intra-zonal embryos were observed.

A 5'-flanking region of embryonic-type myosin heavy chain gene, MYH(M)₇₄₃₋₂, from torafugu Takifugu rubripes regulates developmental muscle-specific expression.

The myosin heavy chain gene, MYH(M)₇₄₃₋₂, is highly expressed in fast muscle fibers of torafugu embryos. However, the regulatory mechanisms involved in its expression have been unclear. In this study, we examined spatio-temporal expression patterns of this gene during development by injecting expression vectors containing the GFP reporter gene fused to the 5'-flanking region of MYH(M)₇₄₃₋₂ into fertilized eggs of zebrafish and medaka.

Electrostatic interactions play a minor role in the binding of ExoS to 14-3-3 proteins.

14-3-3 proteins belong to a family of conserved molecules expressed in all eukaryotic cells that play an important role in a multitude of signalling pathways. 14-3-3 proteins bind either to phosphoserine/phosphothreonine residues or to sequence-specific non-phosphorylated motifs in more than 200 interaction partners [Pozuelo Rubio, Geraghty, Wong, Wood, Campbell, Morrice and Mackintosh (2004) Biochem. J.

Phosphorylation-independent interaction between 14-3-3 and exoenzyme S: from structure to pathogenesis.

14-3-3 proteins are phosphoserine/phosphothreonine-recognizing adapter proteins that regulate the activity of a vast array of targets. There are also examples of 14-3-3 proteins binding their targets via unphosphorylated motifs. Here we present a structural and biological investigation of the phosphorylation-independent interaction between 14-3-3 and exoenzyme S (ExoS), an ADP-ribosyltransferase toxin of Pseudomonas aeruginosa.

Exoenzyme S of Pseudomonas aeruginosa is not able to induce apoptosis when cells express activated proteins, such as Ras or protein kinase B/Akt.

Intracellular targeting of the Pseudomonas aeruginosa toxins, such as exoenzyme S (ExoS), cause cell death, as well as morphological and physiological changes in various tissue culture cells and animal models. In this report we have investigated the mechanism behind ExoS-mediated cell death. In order to address this issue, we have used cell lines expressing activated forms of various components of the Ras signalling pathway in order to evaluate the importance of the Ras pathway for viability and survival upon ExoS infection.

Delineation of exoenzyme S residues that mediate the interaction with 14-3-3 and its biological activity.

14-3-3 proteins belong to a family of conserved molecules expressed in all eukaryotic cells, which play an important role in a multitude of signaling pathways. 14-3-3 proteins bind to phosphoserine/phosphothreonine motifs in a sequence-specific manner. More than 200 14-3-3 binding partners have been found that are involved in cell cycle regulation, apoptosis, stress responses, cell metabolism and malignant transformation.

Mitochondrial ribosomes in a trypanosome.

The nature, and even the existence, of trypanosome mitochondrial ribosomes has been the subject of some debate. We investigated this further in the insect trypanosome, Crithidia fasciculata. In sucrose gradients of parasite lysates, mitochondrial ribosomal RNA co-sediments at approximately 35S with nascent peptides synthesized in the presence of the cytosolic translational inhibitor, cycloheximide.