Naive-like Conversion Overcomes the Limited Differentiation Capacity of Induced Pluripotent Stem Cells

Although induced pluripotent stem (iPS) cells are indistinguishable from ES cells in their expression of pluripotent markers, their differentiation into targeted cells is often limited. Here, we examined whether the limited capacity of iPS cells to differentiate into neural lineage cells could be mitigated by improving their base-line level of pluripotency, i.e. by converting them into the so-called “naive” state. In this study, we used rabbit iPS and ES cells because of the easy availability of both cell types and their typical primed state characters. Repeated passages of the iPS cells permitted their differentiation into early neural cell types (neural stem cells, neurons, and glial astrocytes) with efficiencies similar to ES cells. However, unlike ES cells, their ability to differentiate later into neural cells (oligodendrocytes) was severely compromised. In contrast, after these iPS cells had been converted to a naive-like state, they readily differentiated into mature oligodendrocytes developing characteristic ramified branches, which could not be attained even with ES cells. These results suggest that the naive-like conversion of iPS cells might endow them with a higher differentiation capacity.     

Golgi inheritance in the primitive red alga, Cyanidioschyzon merolae

The Golgi body has important roles in modifying, sorting, and transport of proteins and lipids. Eukaryotic cells have evolved in various ways to inherit the Golgi body from mother to daughter cells, which allows the cells to function properly immediately after mitosis. Here we used Cyanidioschyzon merolae, one of the most suitable systems for studies of organelle dynamics, to investigate the inheritance of the Golgi. Two proteins, Sed5 and Got1, were used as Golgi markers. Using immunofluorescence microscopy, we demonstrated that C. merolae contains one to two Golgi bodies per cell. The Golgi body was localized to the perinuclear region during the G1 and S phases and next to the spindle poles in a microtubule-dependent manner during M phase. It was inherited together with spindle poles upon cytokinesis. These observations suggested that Golgi inheritance is dependent on microtubules in C. merolae.     

Characterization of Site-Specific Mutations in a Short-Chain-Length/Medium-Chain-Length Polyhydroxyalkanoate Synthase: In Vivo and In Vitro Studies of Enzymatic Activity and Substrate Specificity

Saturation point mutagenesis was carried out at position 479 in the polyhydroxyalkanoate (PHA) synthase from Chromobacterium sp. strain USM2 (PhaC_Cs) with specificities for short-chain-length (SCL) [(R)-3-hydroxybutyrate (3HB) and (R)-3-hydroxyvalerate (3HV)] and medium-chain-length (MCL) [(R)-3-hydroxyhexanoate (3HHx)] monomers in an effort to enhance the specificity of the enzyme for 3HHx. A maximum 4-fold increase in 3HHx incorporation and a 1.6-fold increase in PHA biosynthesis, more than the wild-type synthase, was achieved using selected mutant synthases. These increases were subsequently correlated with improved synthase activity and increased preference of PhaC_Cs for 3HHx monomers. We found that substitutions with uncharged residues were beneficial, as they resulted in enhanced PHA production and/or 3HHx incorporation. Further analysis led to postulations that the size and geometry of the substrate-binding pocket are determinants of PHA accumulation, 3HHx fraction, and chain length specificity. In vitro activities for polymerization of 3HV and 3HHx monomers were consistent with in vivo substrate specificities. Ultimately, the preference shown by wild-type and mutant synthases for either SCL (C₄ and C₅) or MCL (C₆) substrates substantiates the fundamental classification of PHA synthases.     

The H19 Imprinting Control Region Mediates Preimplantation Imprinted Methylation of Nearby Sequences in Yeast Artificial Chromosome Transgenic Mice

In the mouse Igf2/H19 imprinted locus, differential methylation of the imprinting control region (H19 ICR) is established during spermatogenesis and is maintained in offspring throughout development. Previously, however, we observed that the paternal H19 ICR, when analyzed in yeast artificial chromosome transgenic mice (YAC-TgM), was preferentially methylated only after fertilization. To identify the DNA sequences that confer methylation imprinting, we divided the H19 ICR into two fragments (1.7 and 1.2 kb), ligated them to both ends of a λ DNA fragment into which CTCF binding sites had been inserted, and analyzed this in YAC-TgM. The maternally inherited λ sequence, normally methylated after implantation in the absence of H19 ICR sequences, became hypomethylated, demonstrating protective activity against methylation within the ICR. Meanwhile, the paternally inherited λ sequence was hypermethylated before implantation only when a 1.7-kb fragment was ligated. Consistently, when two subfragments of the H19 ICR were individually investigated for their activities in YAC-TgM, only the 1.7-kb fragment was capable of introducing paternal allele-specific DNA methylation. These results show that postfertilization methylation imprinting is conferred by a paternal allele-specific methylation activity present in a 1.7-kb DNA fragment of the H19 ICR, while maternal allele-specific activities protect the allele from de novo DNA methylation.     

A recurrent KCNT1 mutation in two sporadic cases with malignant migrating partial seizures in infancy

We performed analysis of KCNT1 in two unrelated patients with malignant migrating partial seizures in infancy. Both patients had intractable focal seizures since two months of age. Their seizures were characterized by a shift of epileptic focus during a single seizure and were resistant to most antiepileptic drugs but responded to vagus nerve stimulation in one and clorazepate in the other. Bidirectional sequencing for KCNT1 was analyzed by standard Sanger sequencing method. A de novo c.862G > A (p.Gly288Ser) missense mutation was identified at the pore region of KCNT1 channel in both patients, whereas all KCNT1 mutations in the previous reports were identified mostly in the intracellular C-terminal region. Computational analysis suggested possible changes in the molecular structure and the ion channel property induced by the Gly288Ser mutation. Because the G-to-A transition was located at CG dinucleotide sequences as previously reported for KCNT1 mutations, the recurrent occurrence of de novo KCNT1 mutations indicated the hot spots of these locations.     

Regulatory effects of Spirulina complex polysaccharides on growth of murine RSV‐M glioma cells through Toll‐like receptor 4

This study is the first to report that Spirulina complex polysaccharides (CPS) suppress glioma growth by down‐regulating angiogenesis via a Toll‐like receptor 4 signal. Murine RSV‐M glioma cells were implanted s.c. into C3H/HeN mice and TLR4 mutant C3H/HeJ mice. Treatment with either Spirulina CPS or Escherichia coli (E. coli) lipopolysaccharides (LPS) strongly suppressed RSV‐M glioma cell growth in C3H/HeN, but not C3H/HeJ, mice. Glioma cells stimulated production of interleukin (IL)‐17 in both C3H/HeN and C3H/HeJ tumor‐bearing mice. Treatment with E. coli LPS induced much greater IL‐17 production in tumor‐bearing C3H/HeN mice than in tumor‐bearing C3H/HeJ mice. In C3H/HeN mice, treatment with Spirulina CPS suppressed growth of re‐transplanted glioma; however, treatment with E. coli LPS did not, suggesting that Spirulina CPS enhance the immune response. Administration of anti‐cluster of differentiation (CD)8, anti‐CD4, anti‐CD8 antibodies, and anti‐asialo GM1 antibodies enhanced tumor growth, suggesting that T cells and natural killer cells or macrophages are involved in suppression of tumor growth by Spirulina CPS. Although anti‐interferon‐γ antibodies had no effect on glioma cell growth, anti‐IL‐17 antibodies administered four days after tumor transplantation suppressed growth similarly to treatment with Spirulina CPS. Less angiogenesis was observed in gliomas from Spirulina CPS‐treated mice than in those from saline‐ or E. coli LPS‐treated mice. These findings suggest that, in C3H/HeN mice, Spirulina CPS antagonize glioma cell growth by down‐regulating angiogenesis, and that this down‐regulation is mediated in part by regulating IL‐17 production.     

Relative growth,morphological sexual maturity,and size of Macrobrachium amazonicum (Heller 1862) (Crustacea,Decapoda, Palaemonidae) in a population with an entirely freshwater life cycle

The objective of this study was to estimate the size of morphological sexual maturity based on the study of relative growth, to determine the maximum size of individuals, and to determine if there are different morphotypes of males in a population of Macrobrachium amazonicum with an entirely freshwater life cycle. Collections were made monthly, with the use of a net, from September 2006 through August 2007. In each individual, the following structures were measured: carapace length (CL, in mm), width of the second pleuron (PlL, mm), length of the carpus (CaL, mm), and length of the propodus (PrL, mm). Relative growth was analyzed by observing the change in growth patterns of certain parts of the body in relation to the independent variable CL. The maximum sizes found were 8.5 and 11.4?mm CL for males and females, respectively. The morphometric variables: length of the carpus (CL?×?CaL) for males, and width of the second pleuron (CL?×?PlL) for females gave the best estimates for the size at maturation, which was 4.26?mm CL for males and 5.39?mm CL for females. The growth pattern in the different stages and the beginning of differential growth seemed to be closely related to reproductive aspects. No indices were found that separated the males into four different morphotypes, as proposed in the literature for coastal or artificially farmed populations. Only the male morphotype termed translucent claw was found in this population. The different morphological patterns in different regions are probably explained by ecological differences in the environments inhabited by these groups, principally in the availability of nutrients and the salinity in which the larvae develop.     

Recruitment of the cohesin loading factor NIPBL to DNA double-strand breaks depends on MDC1, RNF168 and HP1γ in human cells

The cohesin loading factor NIPBL is required for cohesin to associate with chromosomes and plays a role in DNA double-strand break (DSB) repair. Although the NIPBL homolog Scc2 is recruited to an enzymatically generated DSB and promotes cohesin-dependent DSB repair in yeast, the mechanism of the recruitment remains poorly understood. Here we show that the human NIPBL is recruited to the sites of DNA damage generated by micro-irradiation as well as to the sites of DSBs induced by homing endonuclease, I-PpoI. The recruitment of NIPBL was impaired by RNAi-mediated knockdown of MDC1 or RNF168, both of which also accumulate at DSBs. We also show that the recruitment of NIPBL to the sites of DNA damage is mediated by its C-terminal region containing HEAT repeats and Heterochromatin protein 1 (HP1) interacting motif. Furthermore, NIPBL accumulation at damaged sites was also compromised by HP1γ depletion. Taken together, our study reveals that human NIPBL is a novel protein recruited to DSB sites, and the recruitment is controlled by MDC1, RNF168 and HP1γ.     

Extracellular histone induces plasma hyaluronan-binding protein (factor VII activating protease) activation in vivo

Plasma hyaluronan-binding protein (PHBP), an activator of factor VII and prourokinase, is a serine protease circulating as a single-chain proenzyme (pro-PHBP). Pro-PHBP converts to the active two-chain form through autoproteolysis, and effectors that modulate autoactivation can regulate PHBP-mediated processes. Here, we show that histone promotes pro-PHBP autoactivation in vivo. Histone bound to pro-PHBP and promoted intermolecular pro-PHBP binding. Histone-mediated pro-PHBP activation in plasma leads to the formations of bradykinin and PHBP−α₂-antiplasmin complex as well as histone degradation. Pro-PHBP activation was observed in the circulation of mice after injection of histone or lipopolysaccharide, which induced septic response accompanying extracellular histone release. Our results suggest pathophysiological relevance of histone-dependent pro-PHBP activation in hyperinflammatory process.     

Critical role of the nucleolus in activation of the p53-dependent postmitotic checkpoint

Cells eventually exit from mitosis during sustained arrest at the spindle checkpoint, without sister chromatid separation and cytokinesis. The resulting tetraploid cells are arrested in the subsequent G1 phase in a p53-dependent manner by the regulatory function of the postmitotic G1 checkpoint. Here we report how the nucleolus plays a critical role in activation of the postmitotic G1 checkpoint. During mitosis, the nucleolus is disrupted and many nucleolar proteins are translocated from the nucleolus into the cytoplasm. Among the nucleolar factors, Myb-binding protein 1a (MYBBP1A) induces the acetylation and accumulation of p53 by enhancing the interaction between p300 and p53 during prolonged mitosis. MYBBP1A-dependent p53 activation is essential for the postmitotic G1 checkpoint. Thus, our results demonstrate a novel nucleolar function that monitors the prolongation of mitosis and converts its signal into activation of the checkpoint machinery.