A negative correlation between expression profiles of runt-related transcription factor-2 and cystine/glutamate antiporter xCT subunit in ovariectomized mouse bone.

We have previously demonstrated that glutamate (Glu) suppresses cellular proliferation toward self-renewal through a mechanism associated with the depletion of intracellular GSH after promoting the retrograde operation of the bidirectional cystine/Glu antiporter in undifferentiated osteoblastic MC3T3-E1 cells. In this study, we investigated the expression profile of the xCT subunit of the antiporter as well as the master regulator of osteoblastogenesis runt-related transcription factor-2 (Runx2) in ovariectomized mouse bone. In spinal columns isolated 28 days after ovariectomy, a marked reduction was seen with the intensity of Von Kossa staining used as an index of ossification.

Mice lacking Ran binding protein 1 are viable and show male infertility.

The small GTPase Ran plays important roles in multiple aspects of cellular function. Maximal RanGAP activity is achieved with the aid of RanBP1 and/or presumably of RanBP2. Here, we show that RanBP1-knockout mice are unexpectedly viable, and exhibit male infertility due to a spermatogenesis arrest, presumably caused by down-regulation of RanBP2 during spermatogenesis.

Proteomic analysis of importin α-interacting proteins in adult mouse brain.

Many transport factors, such as importins and exportins, have been identified, and the molecular mechanisms underlying nucleocytoplasmic transport have been characterized. The specific molecules that are carried by each transport factor and the temporal profiles that characterize the movements of various proteins into or out of the nucleus, however, have yet to be elucidated. Here, we used a proteomic approach to identify molecules that are transported into the nuclei of adult mouse brain cells via importin α5.

Negative regulation of osteoblastogenesis through downregulation of runt-related transcription factor-2 in osteoblastic MC3T3-E1 cells with stable overexpression of the cystine/glutamate antiporter xCT subunit.

We have previously demonstrated that glutamate (Glu) suppresses cellular proliferation toward self-renewal through a mechanism associated with intracellular GSH depletion mediated by the bidirectional cystine/Glu antiporter in osteoblastic MC3T3-E1 cells cultured in the absence of differentiation inducers. To further evaluate the possible role of the antiporter in osteoblastogenesis, in this study, we have established stable transfectants of the xCT subunit of the antiporter in MC3T3-E1 cells. Stable overexpression led to a significant facilitation of cellular proliferation determined by different indices with increased GSH levels and decreased ROS generation in addition to promoted [(14)C]cystine incorporation, while Glu failed to significantly inhibit cellular proliferation in stable xCT transfectants.

Axotomy induces axonogenesis in hippocampal neurons by a mechanism dependent on importin β.

We characterize the previously unrecognized phenomenon of axotomy-induced axonogenesis in rat embryonic hippocampal neurons in vitro and elucidate the underlying mechanism. New neurites arose from cell bodies after axotomy and grew. These neurites were Tau-1-positive, and the injured axons showed negative immunoreactivity for Tau-1.

Discovery of imidazo[1,2-b]pyridazines as IKKβ inhibitors. Part 2: improvement of potency in vitro and in vivo.

We have increased the potency of imidazo[1,2-b]pyridazine derivatives as IKKβ inhibitors with two strategies. One is to enhance the activity in cell-based assay by adjusting the polarity of molecules to improve permeability. Another is to increase the affinity for IKKβ by the introduction of additional substituents based on the hypothesis derived from an interaction model study.

Exacerbated vulnerability to oxidative stress in astrocytic C6 glioma cells with stable overexpression of the glutamine transporter slc38a1.

We have previously demonstrated the functional expression of glutamine (Gln) transporter (GlnT) believed to predominate in neurons for the neurotransmitter glutamate pool by rat neocortical astrocytes devoid of neuronal marker expression, with exacerbated vulnerability to oxidative stress after transient overexpression. To evaluate molecular mechanisms underlying the exacerbation, we established stable GlnT transfectants in rat astrocytic C6 glioma cells. In two different clones of stable transfectants with increased intracellular Gln levels, exposure to hydrogen peroxide (H(2)O(2)) and A23187, but not to tunicamycin or 2,4-dinitrophenol, led to significant exacerbation of the cytotoxicity compared to cells with empty vector (EV).

Nup358, a nucleoporin, functions as a key determinant of the nuclear pore complex structure remodeling during skeletal myogenesis.

The nuclear pore complex (NPC) is the only gateway for molecular trafficking across the nuclear envelope. The NPC is not merely a static nuclear-cytoplasmic transport gate; the functional analysis of nucleoporins has revealed dynamic features of the NPC in various cellular functions, such as mitotic spindle formation and protein modification. However, it is not known whether the NPC undergoes dynamic changes during biological processes such as cell differentiation.

Gradual downregulation of protein expression of the partner GABA(B)R2 subunit during postnatal brain development in mice defective of GABA(B)R1 subunit.

We have previously shown the functional expression of GABA(B) receptors (GABA(B)R) composed of GABA(B)R1 and GABA(B)R2 subunits with ability to promote proliferation and neuronal differentiation in cultured neural progenitor cells (NPC) isolated from embryonic mouse brains. In this study, we evaluated postnatal changes in the expression profiles of different markers for progenitor, neuronal, astroglial, and microglial cells in brains of GABA(B)R1-null mice. Consistent with undifferentiated murine NPC cultured with epidermal growth factor, a significant and selective decrease was seen in mRNA expression of the proneural gene Mash1 in brains of GABA(B)R1-null mice at 1 day after birth.

Specific monoclonal antibody against the nuclear pore complex protein, Nup96.

Nup96 is a component of the Nup107-160 complex, the largest subunit of the nuclear pore complex. Nup96 is generated as a precursor protein with Nup98. However, the mechanism by which Nup96 contributes to cell function is not clear.

Nuclear import of serum response factor in airway smooth muscle.

We have previously shown that the transcription-promoting activity of serum response factor (SRF) is partially regulated by its extranuclear redistribution. In this study, we examined the cellular mechanisms that facilitate SRF nuclear entry in canine tracheal smooth muscle cells. We used in vitro pull-down assays to determine which karyopherin proteins bound SRF and found that SRF binds KPNA1 and KPNB1 through its nuclear localization sequence.

Paternal mosaicism of an STXBP1 mutation in OS.

Ohtahara syndrome (OS) is one of the most severe and earliest forms of epilepsy. We have recently identified that the de novo mutations of STXBP1 are important causes for OS. Here we report a paternal somatic mosaicism of an STXBP1 mutation.

The timing of retroviral silencing correlates with the quality of induced pluripotent stem cell lines.

Background: Induced pluripotent stem (iPS) cells can be generated from somatic cells by introducing the four transcription factors Oct4, Sox2, Klf4, and c-Myc. Given that iPS cell technology may be useful for medical applications, the quality of iPS cells needs to be maintained during prolonged cultivation. However, it is unclear whether there are any differences in stability among different iPS clones.

Comparing enhancement and washout patterns of hepatic lesions between sonazoid-enhanced ultrasound and contrast-enhanced computed tomography.

Purpose: To compare sonazoid-enhanced ultrasound (SEUS) and contrast-enhanced computed tomography (CECT) enhancement and washout patterns in hepatic lesions.

Methods: Enhancement and washout patterns on SEUS were compared with those on CECT for 61 lesions. There were 36 hepatocellular carcinomas, three intrahepatic cholangiocarcinomas, three metastatic lesions, eight focal nodular hyperplasias, two angiomyolipomas, and nine undetermined benign lesions.

Brain imaging modality before systemic thrombolysis for ischemic stroke within three hours.

Objective: In Japan, MRI-based thrombolysis after CT screening is the most common imaging strategy prior to intravenous thrombolysis (IVT) with tissue plasminogen activator (tPA) within 3 h after ischemic stroke. A choice of MRI with MR angiography (MRA) provides a higher diagnostic accuracy, but may delay an initiation of thrombolysis.

Methods: In our neuro-unit, brain CT is the first screening image for suspected stroke.

Requirement of both NR3A and NR3B subunits for dominant negative properties on Ca2+ mobilization mediated by acquired N-methyl-D-aspartate receptor channels into mitochondria.

Conventional N-methyl-D-aspartate (NMDA) receptor (NMDAR) is a heteromeric complex between the essential NR1 subunit and one of NR2A-D subunits toward functional channels permeable to Ca(2+) rather than Na(+) ions. Although recent studies identified dominant negative NR3A and NR3B subunits, whether these subunits inhibit Ca(2+) mobilization through NMDAR channels into mitochondria is not clarified so far. In this study, we investigated Ca(2+) influx across acquired NMDAR channels composed of different NR subunits artificially expressed in HEK293 cells.

Glutamate preferentially suppresses osteoblastogenesis than adipogenesis through the cystine/glutamate antiporter in mesenchymal stem cells.

We have shown that glutamate (Glu) signaling machineries, such as receptors (GluR) and transporters, are functionally expressed by mesenchymal stem cells, in addition to by their progeny cells such as osteoblasts and chondrocytes. Sustained exposure to Glu induced significant decreases in alkaline phosphatase (ALP) staining and osteoblastic marker gene expression in the mesenchymal C3H10T1/2 stem cells infected with runt-related transcription factor-2 (Runx2) adenovirus, without markedly affecting Oil Red O staining for adipocytes in cells cultured with adipogenic inducers. In cells with Runx2 adenovirus, the cystine/Glu antiporter substrate cystine significantly prevented the decreases by Glu in both ALP staining and osteoblastic marker gene expression, with GluR agonists being ineffective.

Chronic restraint stress impairs neurogenesis and hippocampus-dependent fear memory in mice: possible involvement of a brain-specific transcription factor Npas4.

Neurogenesis in the hippocampus occurs throughout life in a wide range of species and could be associated with hippocampus-dependent learning and memory. Stress is well established to seriously perturb physiological/psychological homeostasis and affect hippocampal function. In the present study, to investigate the effect of chronic restraint stress in early life on hippocampal neurogenesis and hippocampus-dependent memory, 3-week-old mice were subjected to restraint stress 6 days a week for 4 weeks.

Preferential inhibition by antidiarrheic 2-methoxy-4-methylphenol of Ca(2+) influx across acquired N-methyl-D-aspartate receptor channels composed of NR1/NR2B subunit assembly.

In our previous studies, particular phenolic ingredients, such as 2-methoxy-4-methylphenol (2M4MP), of the antidiarrheic drug wood creosote significantly prevented cell death by both hydrogen peroxide and glutamate in cultured rat hippocampal neurons. In this study, we further evaluated the pharmacological properties of 2M4MP on Ca(2+) influx across native and acquired N-methyl-D-aspartate (NMDA) receptor (NMDAR) channels. The addition of 2M4MP significantly prevented the loss of cellular viability and the increase in intracellular free Ca(2+) levels as determined by Fluo-3 in cultured rat hippocampal neurons briefly exposed to NMDA.

Cytoplasmic polyadenylation element-like sequences are involved in dendritic targeting of BDNF mRNA in hippocampal neurons.

Several mRNAs are known to be targeted to dendrites in hippocampal neurons. In this study, we show that brain-derived neurotrophic factor (BDNF) mRNA has two distinct cis-acting dendritic targeting elements in the short 3' untranslated region (UTR): a constitutive element and an activity-dependent one. Moreover, deletion of serial cytoplasmic polyadenylation element (CPE)-like sequences in the short 3'UTR suppressed both constitutive and activity-dependent dendritic targeting.

Effective culture conditions for the induction of pluripotent stem cells.

Background: Induced pluripotent stem (iPS) cells, which are functionally comparable to embryonic stem (ES) cells, can be generated from mouse fibroblasts by expression of a defined set of transcription factors Oct4, Sox2, Klf4, and c-Myc. Since iPS cells are generated from somatic cells, they provide an invaluable source of pluripotent stem cells for cell transplantation therapy that does not present ethical problems. However, the reprogramming efficiency is extremely low, and optimal culture conditions for iPS cell derivation have not been clearly defined.

The mobile FG nucleoporin Nup98 is a cofactor for Crm1-dependent protein export.

Nup98 is a mobile nucleoporin that forms distinct dots in the nucleus, and, although a role for Nup98 in nuclear transport has been suggested, its precise function remains unclear. Here, we show that Nup98 plays an important role in Crm1-mediated nuclear protein export. Nuclear, but not cytoplasmic, dots of EGFP-tagged Nup98 disappeared rapidly after cell treatment with leptomycin B, a specific inhibitor of the nuclear export receptor, Crm1.

Identification of importin alpha1 as a novel constituent of RNA stress granules.

Importin alpha is a nuclear transport receptor well established for its ability to mediate importin beta-mediated nuclear import of proteins that possess classical nuclear localization signal (cNLS). Previously, we reported that importin alpha rapidly accumulates to the nucleus in response to H2O2-induced oxidative stress, which implies a role for this protein in stress response. In this study, we show that importin alpha1 (also known as KPNA2 or Rch1), a major subtype of the importin alpha family, localizes to RNA stress granules (SGs), large cytoplasmic bodies that are thought to function as RNA triage sites during stress response.

Induced tolerance to glutamate neurotoxicity through down-regulation of NR2 subunits of N-methyl-D-aspartate receptors in cultured rat striatal neurons.

We have previously shown differential vulnerabilities to glutamate (Glu) excitotoxicity mediated by the N-methyl-D-aspartate (NMDA) receptor (NMDAR) between rat cortical and rat hippocampal neurons in culture. In this study, we evaluated the possible induced tolerance to NMDA neurotoxicity in cultured rat striatal neurons with prior sustained activation of NMDAR. Brief exposure to Glu or NMDA for 1 hr led to a significant decrease in cellular vitality determined 24 hr later in cultured rat striatal neurons, whereas no marked loss was seen in cellular survival after exposure to Glu or NMDA in striatal neurons previously cultured with Glu or NMDA.

Inhibition by 2-methoxy-4-ethylphenol of Ca2+ influx through acquired and native N-methyl-D-aspartate-receptor channels.

Pharmacological properties were evaluated for the antidiarrheic wood creosote ingredient 2-methoxy-4-ethylphenol (2M4EP), which was shown to be protective against neurotoxicity of N-methyl-D-aspartate (NMDA), to modulate Ca(2+) influx across acquired and native NMDA receptor (NMDAR) channels. NMDA markedly increased intracellular free Ca(2+) levels in HEK293 cells transfected with the expression vector of either NR2A or NR2B subunit together with the essential NR1 subunit vector. Further addition of dizocilpine inhibited the increase by NMDA in intracellular Ca(2+) levels in both types of acquired NMDAR channels, while 2M4EP and the NR2B-subunit-selective antagonist ifenprodil were more effective in inhibiting the increase by NMDA in HEK293 cells expressing NR1/NR2B subunits than in those with NR1/NR2A subunits.