DNA methylation-mediated control of Sry gene expression in mouse gonadal development

DNA methylation at CpG sequences is involved in tissue-specific and developmentally regulated gene expression. The Sry (sex-determining region on the Y chromosome) gene encodes a master protein for initiating testis differentiation in mammals, and its expression is restricted to gonadal somatic cells at 10.5-12.5 days post-coitum (dpc) in the mouse. We found that in vitro methylation of the 5'-flanking region of the Sry gene caused suppression of reporter activity, implying that Sry gene expression could be regulated by DNA methylation-mediated gene silencing. Bisulfite restriction mapping and sodium bisulfite sequencing revealed that the 5'-flanking region of the Sry gene was hypermethylated in the 8.5-dpc embryos in which the Sry gene was not expressed. Importantly, this region was specifically hypomethylated in the gonad at 11.5 dpc, while the hypermethylated status was maintained in tissues that do not express the Sry gene. We concluded that expression of the Sry gene is under the control of an epigenetic mechanism mediated by DNA methylation.     

Isolation of porphyran-degrading marine microorganisms from the surface of red alga, Porphyra yezoensis

Marine microorganisms degrading porphyran (POR) were found on the surface of thalli of Porphyra yezoensis. Fifteen crude microorganism groups softened and liquefied the surface of agar-rich plate medium. Among these, 11 microorganism groups degraded porphyran that consisted of sulfated polysaccharide in Porphyra yezoensis. Following isolation, 7 POR-degradable microorganisms were isolated from the 11 POR-degradable microorganism groups.     

PP2A regulates BCL-2 phosphorylation and proteasome-mediated degradation at the endoplasmic reticulum

Anti-apoptotic activity of BCL-2 is mediated by phosphorylation at the endoplasmic reticulum (ER), but how this phosphorylation is regulated and the mechanism(s) by which it regulates apoptosis are unknown. We purified macromolecular complexes containing BCL-2 from ER membranes and found that BCL-2 co-purified with the main two subunits of the serine/threonine phosphatase, PP2A. The association of endogenous PP2A and BCL-2 at the ER was verified by co-immunoprecipitation and microcystin affinity purification. Knock down or pharmacological inhibition of PP2A caused degradation of phosphorylated BCL-2 and led to an overall reduction in BCL-2 levels. We found that this degradation was due to the action of the proteasome acting selectively at the ER. Conversely, overexpression of PP2A caused elevation in endogenous BCL-2. Most importantly, we found that PP2A knock down sensitized cells to several classes of death stimuli (including ER stress), but this effect was abolished in a genetic background featuring knock in of a non-phosphorylatable BCL-2 allele. These studies support the hypothesis that PP2A-mediated dephosphorylation of BCL-2 is required to protect BCL-2 from proteasome-dependent degradation, affecting resistance to ER stress.     

Expression of foreign type I ribulose-1,5-bisphosphate carboxylase/ oxygenase (EC 4.1.1.39) stimulates photosynthesis in cyanobacterium Synechococcus PCC7942 cells

A reporter gene assay revealed that promoters derived from Synechococcus PCC7942 (S.7942) psbAI and Synechocystis PCC6803 (S.6803) psbAII were suitable for the expression of foreign ribulose-bisphosphate carboxylase (RuBisCO; EC 4.1.1.39) in S.7942 cells. Transformational vectors with a promoter and a foreign RuBisCO gene, cvrbc originated from Allochromatium vinosum, were constructed on a binary vector, pUC303, and introduced to S.7942 cells. When the cvrbc was expressed with the S.7942 psbAI promoter, the total RuBisCO activity increased 2.5- to 4-fold than that of the wild type cell. The S.6803 psbAII promoter increased the activity of the transformant 1.5–2 times of that of wild type cell. There was a significant increase in the rate of photosynthesis depending on the increase of RuBisCO activity. The maximum rate of photosynthesis of the transformant cell was 1.63 times higher than that of the wild type under the illumination of 400 μmol m⁻² s⁻¹, at 20 mM bicarbonate and at 30 °C. Although the photosynthesis of the higher plant is limited by the ability of photosystems under high irradiance and the high CO₂ concentration, that of the S.7942 cell is limited by the RuBisCO activity, even at high CO₂ concentrations and under high irradiance.     

Sex difference in body weight gain and leptin signaling in hypocretin/orexin deficient mouse models

Recent studies in human and animal models of narcolepsy have suggested that obesity in narcolepsy may be due to deficiency of hypocretin signaling, and is also under the influence of environmental factors and the genetic background. In the current study, using two hypocretin/orexin deficient narcoleptic mouse models (i.e. preproorexin knockout (KO) and orexin/ataxin-3 transgenic (TG) mice) with cross-sectional assessments, we have further analyzed factors affecting obesity. We found that both KO and TG narcoleptic mice with mixed genetic backgrounds (N4-5, 93.75-96.88% genetic composition of C57BL/6) tended to be heavier than wild type (WT) mice of 100-200 days old. The body weight of heterozygous mice was intermediate between those of KO and WT mice. Obesity was more prominent in females in both KO and TG narcoleptic mice and was associated with higher serum leptin levels, suggesting a partial leptin resistance. Obesity is less prominent in the congenic TG narcoleptic mice, but is still evident in females. Our results confirmed that hypocretin/orexin ligand deficiency is one of the critical factors for the obese tendency in narcolepsy. However, multiple factors are also likely to affect this phenotype, and a sex difference specific alteration of leptin-hypocretin signaling may be involved.     

CENP-T-W-S-X forms a unique centromeric chromatin structure with a histone-like fold

The multiprotein kinetochore complex must assemble at a specific site on each chromosome to achieve accurate chromosome segregation. Defining the nature of the DNA-protein interactions that specify the position of the kinetochore and provide a scaffold for kinetochore formation remain key goals. Here, we demonstrate that the centromeric histone-fold-containing CENP-T-W and CENP-S-X complexes coassemble to form a stable CENP-T-W-S-X heterotetramer. High-resolution structural analysis of the individual complexes and the heterotetramer reveals similarity to other histone fold-containing complexes including canonical histones within a nucleosome. The CENP-T-W-S-X heterotetramer binds to and supercoils DNA. Mutants designed to compromise heterotetramerization or the DNA-protein contacts around the heterotetramer strongly reduce the DNA binding and supercoiling activities in?vitro and compromise kinetochore assembly in?vivo. These data suggest that the CENP-T-W-S-X complex forms a unique nucleosome-like structure to generate contacts with DNA, extending the "histone code" beyond canonical nucleosome proteins.     

In Vitro Modeling of Paraxial Mesodermal Progenitors Derived from Induced Pluripotent Stem Cells

Induced pluripotent stem (iPS) cells are generated from adult somatic cells by transduction of defined factors. Given their unlimited proliferation and differentiation potential, iPS cells represent promising sources for cell therapy and tools for research and drug discovery. However, systems for the directional differentiation of iPS cells toward paraxial mesodermal lineages have not been reported. In the present study, we established a protocol for the differentiation of mouse iPS cells into paraxial mesodermal lineages in serum-free culture. The protocol was dependent on Activin signaling in addition to BMP and Wnt signaling which were previously shown to be effective for mouse ES cell differentiation. Independently of the cell origin, the number of transgenes, or the type of vectors used to generate iPS cells, the use of serum-free monolayer culture stimulated with a combination of BMP4, Activin A, and LiCl enabled preferential promotion of mouse iPS cells to a PDGFR-α⁺/Flk-1⁻ population, which represents a paraxial mesodermal lineage. The mouse iPS cell-derived paraxial mesodermal cells exhibited differentiation potential into osteogenic, chondrogenic, and myogenic cells both in vitro and in vivo and contributed to muscle regeneration. Moreover, purification of the PDGFR-α⁺/KDR⁻ population after differentiation allowed enrichment of human iPS cell populations with paraxial mesodermal characteristics. The resultant PDGFR-α⁺/KDR⁻ population derived from human iPS cells specifically exhibited osteogenic, chondrogenic, and myogenic differentiation potential in vitro, implying generation of paraxial mesodermal progenitors similar to mouse iPS cell-derived progenitors. These findings highlight the potential of protocols based on the serum-free, stepwise induction and purification of paraxial mesodermal cell lineages for use in stem cell therapies to treat diseased bone, cartilage, and muscle.     

Affixin activates Rac1 via betaPIX in C2C12 myoblast

Affixin/beta-parvin is an integrin-linked kinase (ILK)-binding focal adhesion protein highly expressed in skeletal muscle and heart. To elucidate the possible role of affixin in skeletal muscle, we established stable C2C12 cell line expressing T7-tagged human affixin (C2C12-affixin cells). Exogenous expression of affixin promotes lamellipodium formation where affixin, ILK alphap21-activated kinase (PAK)-interactive exchange factor (PIX) and betaPIX accumulate. The association of affixin and betaPIX was confirmed by immunoprecipitation and pull down assay. In C2C12-affixin cells, an increased level of activated Rac1 but not Cdc42 was observed, and mutant betaPIX lacking guanine nucleotide exchange factor activity inhibited lamellipodium formation. These results suggest that affixin is involved in reorganization of subsarcolemmal cytoskeletal actin by activation of Rac1 through alpha and betaPIXs in skeletal muscle.     

Temperature dependence of seedling establishment of a perennial, Dioscorea tokoro

The temperature dependence of seed germination and seedling growth was analyzed in Dioscorea tokoro, an East Asian summer-green perennial. Seeds were able to germinate fully only at 11°–20°C. At around 17°–20°C the first leaf petiole of the seedling elongated and quickly set the first leaf blade at a position enabling photosynthesis. At temperatures higher than 20°C petiole elongation was retarded, and seedlings formed a rhizome and established as a perennial. The rhizome size increased with temperature up to 29°C. Thus, during growth immediately after germination, temperature appears to be a key factor in determining whether the plant establishes as a perennial or grows rapidly without rhizome thickening. Received: April 6, 2001 / Accepted: September 14, 2001