Aberrant gene expression and sexually incompatible genomic imprinting in oocytes derived from XY mouse embryonic stem cells in vitro.

Mouse embryonic stem cells (ESCs) have the potential to differentiate into germ cells (GCs) in vivo and in vitro. Interestingly, XY ESCs can give rise to both male and female GCs in culture, irrespective of the genetic sex. Recent studies showed that ESC-derived primordial GCs contributed to functional gametogenesis in vivo; however, in vitro differentiation techniques have never succeeded in generating mature oocytes from ESCs due to cryptogenic growth arrest during the preantral follicle stages of development.

Functional mechanisms and roles of adaptor proteins in abl-regulated cytoskeletal actin dynamics.

Abl is a nonreceptor tyrosine kinase and plays an essential role in the modeling and remodeling of F-actin by transducing extracellular signals. Abl and its paralog, Arg, are unique among the tyrosine kinase family in that they contain an unusual extended C-terminal half consisting of multiple functional domains. This structural characteristic may underlie the role of Abl as a mediator of upstream signals to downstream signaling machineries involved in actin dynamics.

Siglec-15 protein regulates formation of functional osteoclasts in concert with DNAX-activating protein of 12 kDa (DAP12).

Osteoclasts are multinucleated giant cells that reside in osseous tissues and resorb bone. Signaling mediated by receptor activator of nuclear factor (NF)-κB (RANK) and its ligand leads to the nuclear factor of activated T cells 2/c1 (NFAT2 or NFATc1) expression, a critical step in the formation of functional osteoclasts. In addition, adaptor proteins harboring immunoreceptor tyrosine-based activation motifs, such as DNAX-activating protein of 12 kDa (DAP12), play essential roles.

Advantages and potential of lipid-membrane-incorporating fullerenes prepared by the fullerene-exchange method.

Lipid-membrane-incorporating C(60) and C(70) (LMIC(60) and LMIC(70)) were prepared by the fullerene-exchange reaction from the γ-cyclodextrin cavity to vesicles (we call this method the "exchange method"). An advantage of this method is that the ratios of [C(60)]/[lipids] and [C(70)]/[lipids] can be arbitrarily controlled by adjusting the ratios of the fullerenes and liposome. The maximum ratio (30 mol%) obtained was approximately 14 and 100 times higher than those achieved for LMIC(60) and LMIC(70) , respectively, that were prepared by the classical method, which we call the "premixing method" (dissolving lipids and C(60) or C(70) in chloroform, followed by concentration and extraction with water).

Identification of SAMT family proteins as substrates of MARCH11 in mouse spermatids.

MARCH11, a RING-finger transmembrane ubiquitin ligase, is predominantly expressed in spermatids and localized to the trans-Golgi network (TGN) and multivesicular bodies (MVBs). Because ubiquitination acts as a sorting signal of cargo proteins, MARCH11 has been postulated to mediate selective protein sorting via the TGN-MVB pathway. However, the physiological substrate of MARCH11 has not been identified.

Abl-1-bridged tyrosine phosphorylation of VASP by Abelson kinase impairs association of VASP to focal adhesions and regulates leukaemic cell adhesion.

Mena [mammalian Ena (Enabled)]/VASP (vasodilator-stimulated phosphoprotein) proteins are the homologues of Drosophila Ena. In Drosophila, Ena is a substrate of the tyrosine kinase DAbl (Drosophila Abl). However, the link between Abl and the Mena/VASP family is not fully understood in mammals.

The l-Ser analog #290 promotes bone recovery in OP and RA mice.

We previously characterized the l-Ser analog #290, H(tBut)-l-Ser-O-Methyl·HCl, as a novel inhibitor of osteoclastogenesis which functions in both mouse and human cells. Here, we assessed the activity of #290 in animal models of osteoporosis and rheumatoid arthritis. Treatment of animals with #290 both prevented bone loss and led to the recovery of lost bone in osteoporotic mice.

Identification and functional analysis of a new phosphorylation site (Y398) in the SH3 domain of Abi-1.

Abi-1 is an adaptor protein for Abelson kinase (c-Abl), and Abi-1 promotes the Abl-mediated phosphorylation of Mammalian Enabled (Mena) by binding both c-Abl and Mena. Here, we identified a new phosphorylation site (Y398) in the SH3 domain of Abi-1, and disruption of Y398, combined with the previously identified phosphorylation site Y213, significantly weakens the binding of Abi-1 to c-Abl. The SH3 domain of Abi-1 and the proline-rich domain of c-Abl are involved in this interaction.

Acid sphingomyelinase regulates osteoclastogenesis by modulating sphingosine kinases downstream of RANKL signaling.

Acid sphingomyelinase (ASM) was identified as a gene induced by NFAT2 activation in osteoclasts. Suppression of ASM expression in bone marrow macrophages by knockdown enhanced c-Fos/NFAT2 expression, increasing the number of TRAP-positive multinucleated cells in vitro. SphK1 was upregulated during the late stage of osteoclastogenesis, while SphK2 expression remained constant.

Photodynamic Activity of Liposomal Photosensitizers via Energy Transfer from Antenna Molecules to [60]Fullerene.

Photodynamic therapy (PDT) is an emerging approach for the treatment of tumor diseases that has received growing interest in the past few years. In this study, we constructed liposomal photosensitizers (PS) for PDT by shoehorning as light-harvesting "antenna" molecules and dense [60]fullerene (C60) into lipid membrane bilayers. The liposomal PS showed improved photodynamic activity toward human cancer cells via the photoenergy transfer from photoactivated antenna molecules to C60.

Potential involvement of Twist2 and Erk in the regulation of osteoblastogenesis by HB-EGF-EGFR signaling.

Epidermal growth factor (EGF) family members play important roles in the skeletal system. In this study, we examined the role of EGF receptor (EGFR) signaling in osteoblastogenesis in vitro. The expression of HB-EGF and epiregulin (EPR) was transiently induced within 24 h after osteogenic stimulation, but when preosteoblastic MC3T3-E1 cells were incubated with HB-EGF or EPR, osteoblast differentiation was inhibited.

Identification of a novel L-serine analog that suppresses osteoclastogenesis in vitro and bone turnover in vivo.

Osteoclasts are multinucleated giant cells with bone resorbing activity. We previously reported that the expression of the transcription factor NFAT2 (NFATc1) induced by receptor activator of NF-kappaB ligand (RANKL) is essential for the formation of multinucleated cells. We subsequently identified L-Ser in the differentiation medium as necessary for the expression of NFAT2.

Activator of G protein signaling 8 (AGS8) is required for hypoxia-induced apoptosis of cardiomyocytes: role of G betagamma and connexin 43 (CX43).

Ischemic injury of the heart is associated with activation of multiple signal transduction systems including the heterotrimeric G-protein system. Here, we report a role of the ischemia-inducible regulator of G betagamma subunit, AGS8, in survival of cardiomyocytes under hypoxia. Cultured rat neonatal cardiomyocytes (NCM) were exposed to hypoxia or hypoxia/reoxygenation following transfection of AGS8siRNA or pcDNA::AGS8.

Direct and short-time uptake of [70]fullerene into the cell membrane using an exchange reaction from a [70]fullerene-gamma-cyclodextrin complex and the resulting photodynamic activity.

[70]Fullerene (C70) was directly incorporated into the cell membrane using an exchange reaction from a C70-gamma-cyclodextrin (gamma-CDx) complex within 10 min and the incorporated C70 acted as a photodynamic sensitiser for a cancer cell.

Photodynamic activity of C70 caged within surface-cross-linked liposomes.

[70]Fullerene (C(70)) encapsulated into a surface-cross-linked liposome, a so-called cerasome, was prepared by an exchange reaction incorporating C(70)gamma-cyclodextrin complexes into lipid membranes. Fullerene exchange in a cerasome-incorporated C(70) (CIC(70)), as well as in a lipid-membrane-incorporated C(70) (LMIC(70)), was completed within 1 min with stirring at 25 degrees C. CIC(70) was more resistant to lysis than LMIC(70) towards lysing agents such as surfactants.

Intracellular uptake and photodynamic activity of water-soluble [60]- and [70]fullerenes incorporated in liposomes.

Water-soluble fullerenes have attracted attention as promising compounds that have been used to forge new paths in the field of photo-biochemistry. To prepare water-soluble fullerenes, we employed lipid-membrane-incorporated fullerenes (LMICx; x=60 or 70) by using the fullerene exchange method from a gamma-cyclodextrin (gamma-CD) cavity to vesicles. LMIC60 have low toxicity in the dark and engender cell death by photoirradiation (lambda>350 nm).

Solubilisation of [60]fullerenes using block copolymers and evaluation of their photodynamic activities.

Unmodified [60]fullerenes (C60) were solubilised with high stability using various type of poly(ethylene glycol) (PEG) based block copolymer micelles. Block copolymer micelle-incorporated C60 fullerenes were studied in cultures for biological activities using human cervical cancer HeLa cells. As a result, the cationic block copolymer micelles delivered C60 into the cells depending on their surface densities and showed cytotoxicity under photoirradiation.

Disruption of c-Jun reduces cellular migration and invasion through inhibition of c-Src and hyperactivation of ROCK II kinase.

The spread of metastatic tumors to different organs is associated with poor prognosis. The metastatic process requires migration and cellular invasion. The protooncogene c-jun encodes the founding member of the activator protein-1 family and is required for cellular proliferation and DNA synthesis in response to oncogenic signals and plays an essential role in chemical carcinogenesis.

Identification of angiogenin as the osteoclastic bone resorption-inhibitory factor in bovine milk.

We identified, for the first time, the factor responsible for inhibiting osteoclast-mediated bone resorption in the basic protein fraction of bovine milk (milk basic protein, MBP). The protein was purified by a combination of ion and gel column chromatography from MBP, based on its activity to prevent unfractionated rabbit bone cells from forming pits on dentine slices. It was found to have a molecular weight of 15 kDa on SDS-PAGE, and the sequence of the N-terminal 25 amino acid residues was identical to that of bovine angiogenin.

Induction of cell death by photodynamic therapy with water-soluble lipid-membrane-incorporated [60]fullerene.

Intracellular uptake of a lipid-membrane-incorporated C(60) with a cationic surface into HeLa cells was found to induce cell death under visible light irradiation in high efficiency.

A novel role of L-serine (L-Ser) for the expression of nuclear factor of activated T cells (NFAT)2 in receptor activator of nuclear factor kappa B ligand (RANKL)-induced osteoclastogenesis in vitro.

Multinucleated cell formation is crucial for osteoclastogenesis, and the expression of nuclear factor of activated T cells (NFAT)2 (NFATc1) is essential for this process. We previously found, using mouse RAW264 cells, that culture at high cell density blocked progression to the multinucleated cell stage induced by stimulation with receptor activator of nuclear factor kappaB ligand (RANKL). Here, we have confirmed this finding in a bone marrow cell system and extended the analysis further.

A rapid protein expression and purification system using Chinese hamster ovary cells expressing retrovirus receptor.

Conventional stable protein expression systems using mammalian cells include a time-consuming step of antibiotic resistance-based cell cloning. Here, we report a rapid flow cytometry-based method for the collection of retrovirus vector-infected Chinese hamster ovary (CHO) cells that express desired proteins. The vector carries the genes for green fluorescent protein (GFP), as a marker, and glutathione-S-transferase (GST), to express the desired protein as a GST-fusion construct.

Src homology 2 (SH2)-containing 5'-inositol phosphatase localizes to podosomes, and the SH2 domain is implicated in the attenuation of bone resorption in osteoclasts.

c-Src plays an important role in bone resorption by osteoclasts. Here, we show using wild-type and ship(-/-) osteoclasts that Src homology 2 (SH2)-containing 5'-inositol phosphatase (SHIP) appeared to negatively regulate bone resorption activated by c-Src. SHIP was found to localize to podosomes under the influence of c-Src, and the presence of either the amino-terminal region comprising the SH2 domain or the carboxyl-terminal region was sufficient for its localization.