Maturation/M-phase-promoting factor is the universal inducer of M-phase in eukaryotic cells. It is currently accepted that M-phase-promoting factor is identical to the kinase cyclin B–Cdk1. Here we show that cyclin B–Cdk1 and M-phase-promoting factor are not in fact synonymous. Instead, M-phase-promoting factor contains at least two essential components: cyclin B–Cdk1 and another kinase, Greatwall kinase. In the absence of Greatwall kinase, the M-phase-promoting factor is undetectable in oocyte cytoplasm even though cyclin B–Cdk1 is fully active, whereas M-phase-promoting factor activity is restored when Greatwall kinase is added back. Although the excess amount of cyclin B–Cdk1 alone, but not Greatwall kinase alone, can induce nuclear envelope breakdown, spindle assembly is abortive. Addition of Greatwall kinase greatly reduces the amount of cyclin B–Cdk1 required for nuclear envelope breakdown, resulting in formation of the spindle with aligned chromosomes. M-phase-promoting factor is thus a system consisting of one kinase (cyclin B–Cdk1) that directs mitotic entry and a second kinase (Greatwall kinase) that suppresses the protein phosphatase 2A-B55 which opposes cyclin B–Cdk1.

Background-It has been proposed that bone marrow-derived cells infiltrate the neointima, where they differentiate into smooth muscle (SM) cells; however, technical limitations have hindered clear identification of the lineages of bone marrow-derived "SM cell-like" cells. Methods and Results-Using a specific antibody against the definitive SM cell lineage marker SM myosin heavy chain (SM-MHC) and mouse lines in which reporter genes were driven by regulatory programs for either SM-MHC or SM ␣-actin, we demonstrated that although some bone marrow-derived cells express SM ␣-actin in the wire injury-induced neointima, those cells did not express SM-MHC, even 30 weeks after injury. Likewise, no SM-MHC ϩ bone marrow-derived cells were found in vascular lesions in apolipoprotein E

Wnt proteins direct embryonic patterning, but the regulatory basis of their distribution and signal reception remain unclear. Here, we show that endogenous Wnt8 protein is distributed in a graded manner in Xenopus embryo and accumulated on the cell surface in a punctate manner in association with “N-sulfo-rich heparan sulfate (HS),” not with “N-acetyl-rich HS”. These two types of HS are differentially clustered by attaching to different glypicans as core proteins. N-sulfo-rich HS is frequently internalized and associated with the signaling vesicle, known as the Frizzled/Wnt/LRP6 signalosome, in the presence of Wnt8. Conversely, N-acetyl-rich HS is rarely internalized and accumulates Frzb, a secreted Wnt antagonist. Upon interaction with Frzb, Wnt8 associates with N-acetyl-rich HS, suggesting that N-acetyl-rich HS supports Frzb-mediated antagonism by sequestering Wnt8 from N-sulfo-rich HS. Thus, these two types of HS clusters may constitute a cellular platform for the distribution and signaling of Wnt8.

From whole genome sequencing of an allotetraploid frog, Xenopus laevis, two homeologous sets (L and S) of four Hox clusters A through D (HoxA.L/S, HoxB.L/S, HoxC.L/S, and HoxD.L/S) and 13 paralogous groups (PGs) with 76 genes were identified, allowing us to carry out the first comprehensive analyses of hox gene expression in vertebrates. Expression of all hox genes during development and in adult tissues was analyzed by RNA-sequencing. The expression levels of most hox genes were similar between homeologs, but in some pairs, large differences were observed and several of these were confirmed by RT-PCR and whole mount in situ hybridization experiments. These results indicate that subfunctionalization of hox genes may have occurred since allotetraploidization. Furthermore, comprehensive analysis of hox gene expression during early development did not agree with the hypothesis of temporal collinearity especially in genes belonging to PG2 to PG10.

Aims/hypothesis. Type 1 diabetes mellitus, a T-cellmediated autoimmune disease, results from the selective destruction of insulin-producing pancreatic beta cells. Autoantibodies against beta-cell components are used clinically as sensitive markers of this disease; however, their physiological role has not been clear. To investigate the role of glutamic acid decarboxylase 65 (GAD65) in the development of the Type 1 diabetes of non-obese diabetic (NOD) mice, we analysed and characterised NOD mice with targeted disruption of the GAD65 gene. Methods. GAD65-deficient mice were previously established. After backcrossing the knockout mutation onto the NOD genetic background for up to eight generations, female littermates of the three resulting genotypes were produced by intercrossing: GAD65 +/+ (n=23), GAD65 +/− (n=62), and GAD65 −/− (n=31).Results. The cumulative incidence of autoimmune diabetes showed no significant difference among the three groups in longitudinal studies using the KaplanMeier method. Islet morphology showed that the progression of islet infiltration did not differ significantly between the three groups. Conclusion/interpretation. The cumulative incidence of autoimmune diabetes was not influenced by the GAD65 deficiency. These data suggest that GAD65 is not a major regulatory target of beta-cell autoimmunity in NOD mice. [Diabetologia (2004) . It is, however, unclear whether the anti-GAD antibodies participate directly in betacell destruction or arise secondarily in response to the release of autoantigens from islets damaged by other components of the immune system. To investigate the role of GAD65 in the development of the autoimmune diabetes of NOD mice, we have generated GAD65 −/− NOD mice and analysed the onset and incidence of diabetes in these mice. Materials and methodsGeneration and genotyping of a GAD65 mutant mouse. We previously established 129/Ola-derived embryonic stem-cell clones carrying the GAD65 targeted mutation through homologous recombination [2]. GAD65 knockout embryonic stem cells were injected into C57BL/6 mouse blastocysts to make chimeras, and germ-line transmission was achieved. GAD65

Antibody-dependent enhancement (ADE) of influenza A NWS virus infection was investigated in primary murine macrophages (M phi) using anti-hemagglutinin(HA) monoclonal antibody (mAB). Contrary to previous reports of abortive influenza virus infection in primary M phi, this study demonstrated that the NWS virus replicated productively in both resident peritoneal M phi and thioglycolate-elicited peritoneal M phi providing cleavage of the HA was achieved by trypsin; 5 micrograms/ml of trypsin was the optimum concentration for the induction of infectivity. Under multiple-cycle growth conditions in the presence of mAB at various concentrations in trypsin-containing media, ADE was demonstrated in both M phi in the presence of subneutralizing concentrations of mAB. Flow cytometric analysis showed that the mechanism of virus entry into M phi could be through HA to specific virus receptors, or HA plus antibody to Fc receptors. These results indicate that ADE of the NWS virus infection actually occurs on Fc receptor-bearing primary murine M phi depending on the concentration of antibody in the presence of the appropriate protease for cleavage of viral HA.

BackgroundUnderstanding the general trends in developmental changes during animal evolution, which are often associated with morphological diversification, has long been a central issue in evolutionary developmental biology. Recent comparative transcriptomic studies revealed that gene expression profiles of mid-embryonic period tend to be more evolutionarily conserved than those in earlier or later periods. While the hourglass-like divergence of developmental processes has been demonstrated in a variety of animal groups such as vertebrates, arthropods, and nematodes, the exact mechanism leading to this mid-embryonic conservation remains to be clarified. One possibility is that the mid-embryonic period (pharyngula period in vertebrates) is highly prone to embryonic lethality, and the resulting negative selections lead to evolutionary conservation of this phase. Here, we tested this “mid-embryonic lethality hypothesis” by measuring the rate of lethal phenotypes of three different species of vertebrate embryos subjected to two kinds of perturbations: transient perturbations and genetic mutations.ResultsBy subjecting zebrafish (Danio rerio), African clawed frog (Xenopus laevis), and chicken (Gallus gallus) embryos to transient perturbations, namely heat shock and inhibitor treatments during three developmental periods [early (represented by blastula and gastrula), pharyngula, and late], we found that the early stages showed the highest rate of lethal phenotypes in all three species. This result was corroborated by perturbation with genetic mutations. By tracking the survival rate of wild-type embryos and embryos with genetic mutations induced by UV irradiation in zebrafish and African clawed frogs, we found that the highest decrease in survival rate was at the early stages particularly around gastrulation in both these species.ConclusionIn opposition to the “mid-embryonic lethality hypothesis,” our results consistently showed that the stage with the highest lethality was not around the conserved pharyngula period, but rather around the early period in all the vertebrate species tested. These results suggest that negative selection by embryonic lethality could not explain hourglass-like conservation of animal embryos. This highlights the potential contribution of alternative mechanisms such as the diversifying effect of positive selections against earlier and later stages, and developmental constraints which lead to conservation of mid-embryonic stages.Electronic supplementary materialThe online version of this article (10.1186/s13227-018-0095-0) contains supplementary material, which is available to authorized users.

Spin state transition and intermetallic charge transfer can essentially change material structural and physical properties with a fashion of excluding external chemical doping. However, these two effects have rarely been found to occur sequentially in a specific material. In this article, we show the realization of these two phenomena in a perovskite oxide PbCoO3 with a simple ABO3 composition under high pressure. PbCoO3 possesses a peculiar A-and B-site ordered charge distribution Pb 2+ Pb 4+ 3Co 2+ 2Co 3+ 2O12 with insulating behavior at ambient conditions. The high spin Co 2+ gradually changes to low spin with increasing pressure up to about 15 GPa, leading to the anomalous increase of resistance magnitude. Between 15 GPa and 30 GPa, the intermetallic charge transfer occurs between Pb 4+ and Co 2+ cations. The accumulated charge-transfer effect triggers a metal-insulator transition as well as a first-order structural phase transition toward a Tetra.-I phase at the onset of ~20 GPa near room temperature. On further compression over 30 GPa, the charge transfer completes, giving rise to another first-order structural transformation toward a Tetra.-II phase and the reentrant electrical insulating behavior.