Cytoplasmic asters are required for progression past the first cell cycle in cloned mouse embryos

Unlike the oocytes of most other animal species, unfertilized murine oocytes contain cytoplasmic asters, which act as microtubule-organizing centers following fertilization. This study examined the role of asters during the first cell cycle of mouse nuclear transfer (NT) embryos. NT was performed by intracytoplasmic injection of cumulus cells. Cytoplasmic asters were localized by staining with an anti-alpha-tubulin antibody. Enucleation of MII oocytes caused no significant change in the number of cytoplasmic asters. The number of asters decreased after transfer of the donor nuclei into these enucleated oocytes, probably because some of the asters participated in the formation of the spindle that anchors the donor chromosomes. The cytoplasmic asters became undetectable within 2 h of oocyte activation, irrespective of the presence or absence of the donor chromosomes. After the standard NT protocol, a spindle-like structure persisted between the pseudopronuclei of these oocytes throughout the pronuclear stage. The asters reappeared shortly before the first mitosis and formed the mitotic spindle. When the donor nucleus was transferred into preactivated oocytes (delayed NT) that were devoid of free asters, the microtubules and microfilaments were distributed irregularly in the ooplasm and formed dense bundles within the cytoplasm. Thereafter, all of the delayed NT oocytes underwent fragmentation and arrested development. Treatment of these delayed NT oocytes with Taxol, which is a microtubule-assembling agent, resulted in the formation of several aster-like structures and reduced fragmentation. Some Taxol-treated oocytes completed the first cell cycle and developed further. This study demonstrates that cytoplasmic asters play a crucial role during the first cell cycle of murine NT embryos. Therefore, in mouse NT, the use of MII oocytes as recipients is essential, not only for chromatin reprogramming as previously reported, but also for normal cytoskeletal organization in reconstructed oocytes.     

Stereoselectivity in deoxygenation of 5-hydroxy-5-phosphinyl-hexofuranoses (alpha-hydroxyphosphonates)

The addition of dimethyl phosphonate to six different hexofuranos-5-uloses in the presence of DBU, followed by esterification with methoxalyl chloride and then radical reduction, afforded 5-deoxy-5-dimethoxyphosphinyl-D- and L-hexofuranoses. The stereoselectivity of the deoxygenation and possible transition-state models are discussed.     

Defective smooth muscle development in qkI-deficient mice

The qkI gene encodes an RNA binding protein which was identified as a candidate for the classical neurologic mutation, qkv. Although qkI is involved in glial cell differentiation in mice, qkI homologues in other species play important roles in various developmental processes. Here, we show a novel function of qkI in smooth muscle cell differentiation during embryonic blood vessel formation. qkI null embryos died between embryonic day 9.5 and 10.5. Embryonic day 9.5 qkI null embryos showed a lack of large vitelline vessels in the yolk sacs, kinky neural tubes, pericardial effusion, open neural tubes and incomplete embryonic turning. Using X-gal and immunohistochemical staining, qkI is first shown to be expressed in endothelial cells and smooth muscle cells. Analyses of qkI null embryos in vivo and in vitro revealed that the vitelline artery was too thin to connect properly to the yolk sac, thereby preventing remodeling of the yolk sac vasculature, and that the vitelline vessel was deficient in smooth muscle cells. Addition of QKI and platelet-endothelial cell adhesion molecule-1 positive cells to an in vitro para-aortic splanchnopleural culture of qkI null embryos rescued the vascular remodeling deficit. These data suggest that QKI protein has a critical regulatory role in smooth muscle cell development, and that smooth muscle cells play an important role in inducing vascular remodeling.     

Molecular cloning and characterization of TPP36 and its isoform TPP32, novel substrates of Abl tyrosine kinase

We have molecularly cloned TPP36, a novel 36 kDa protein with 281 amino acids that was identified as a protein phosphorylated in B progenitor cells following stimulation with pervanadate/H(2)O(2). Analysis with anti-TPP36 antiserum revealed that TPP36 was expressed ubiquitously and had an isoform with 236 amino acids, designated TPP32. TPP36/32 were localized mainly in cytoplasm despite the presence of a typical nuclear localization signal sequence. These proteins were phosphorylated preferentially by Abl among a panel of tyrosine kinases examined. Phosphorylation of tyrosine 120 in TPP36/32 led to an apparent mobility shift in sodium dodecyl sulfate-polyacrylamide gel electrophoresis, suggesting conformational change in the phosphorylated protein. Thus, TPP36/32 appear to be novel substrates of Abl tyrosine kinase.     

Daxx enhances Fas-mediated apoptosis in a murine pro-B cell line,BAF3

Daxx has been shown to play an essential in type I interferon (IFN-/β)-mediated suppression of B cell development and apoptosis. Recently, we demonstrated that Tyk2 is directly involved in IFN signaling for the induction and nuclear translocation of Daxx, which may result in growth arrest and/or apoptosis of B lymphocyte progenitors. To clarify the mechanism of Daxx-mediated apoptosis signaling in B lymphocyte progenitors, here we introduced an efficient suicide switch in a murine pro-B cell line, BAF3, by expressing FK506-binding protein-fused Fas intracellular domain (FKBP-Fas) and Daxx. It allows us to monitor Fas/Daxx-mediated signal by induction of Fas dimerization with the dimerizer drug AP20187. AP20187-mediated Fas dimerization induced not only apoptosis but also Jun N-terminal kinase (JNK) activation. However, AP20187 had no effect on cells expressing either Fas or Daxx only. Furthermore, expression of a JNK inhibitor, the JNK-binding domain of JIP-1, resulted in resistance to AP20187-mediated apoptosis in cells expressing FKBP-Fas and Daxx. These results imply that our novel suicide switch system may provide a powerful tool to delineate or identify the signaling molecules for Daxx-mediated apoptotic machinery in B lymphocyte progenitors through JNK activation.     

T-0901317, a synthetic liver X receptor ligand,inhibits development of atherosclerosis in LDL receptor-deficient mice

Liver X receptors (LXR alpha and LXR beta) are nuclear receptors, which are important regulators of cholesterol and lipid metabolism. LXRs control genes involved in cholesterol efflux in macrophages, bile acid synthesis in liver and intestinal cholesterol absorption. LXRs also regulate genes participating in lipogenesis. To determine whether the activation of LXR promotes or inhibits development of atherosclerosis, T-0901317, a synthetic LXR ligand, was administered to low density lipoprotein receptor (LDLR)(-/-) mice. T-0901317 significantly reduced the atherosclerotic lesions in LDLR(-/-) mice without affecting plasma total cholesterol levels. This anti-atherogenic effect correlated with the plasma concentration of T-0901317, but not with high density lipoprotein cholesterol, which was increased by T-0901317. In addition, we observed that T-0901317 increased expression of ATP binding cassette A1 in the lesions in LDLR(-/-) mice as well as in mouse peritoneal macrophages. T-0901317 also significantly induced cholesterol efflux activity in peritoneal macrophages. These results suggest that LXR ligands may be useful therapeutic agents for the treatment of atherosclerosis.     

Dynamic structure of pharaonis phoborhodopsin (sensory rhodopsin II) and complex with a cognate truncated transducer as revealed by site-directed 13C solid-state NMR

We have recorded (13)C nuclear magnetic resonance (NMR) spectra of [3-(13)C]Ala, [1-(13)C]Val-labeled pharaonis phoborhodopsin (ppR or sensory rhodopsin II) incorporated into egg PC (phosphatidylcholine) bilayer, by means of site-directed high-resolution solid-state NMR techniques. Seven (13)C NMR signals from transmembrane alpha-helices were resolved for [3-(13)C]Ala-ppR at almost the same positions as those of bacteriorhodopsin (bR), except for the suppressed peaks in the loop regions in spite of the presence of at least three Ala residues. In contrast, (13)C NMR signals from the loops were visible from [1-(13)C]Val-ppR but their peak positions of the transmembrane alpha-helices are not always the same between ppR and bR. The motional frequency of the loop regions in ppR was estimated as 10(5) Hz in view of the suppressed peaks from [3-(13)C]Ala-ppR due to interference with proton decoupling frequency. We found that conformation and dynamics of ppR were appreciably altered by complex formation with a cognate truncated transducer pHtr II (1-159). In particular, the C-terminal alpha-helix protruding from the membrane surface is involved in the complex formation and subsequent fluctuation frequency is reduced by one order of magnitude.