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Guanine-rich DNA and RNA can form a four-stranded structure, termed G-quadruplexes (G4s) in vitro as well as in cells. The formation of G4 is implicated in many physiological events, such as gene transcription, translation, and epigenetics. However, the presence of G4 has not been revealed in the brain. Here, we demonstrate the localization of G4 in the mouse brain by immunohistochemical analysis. In cultured mouse forebrain neurons, numerous punctate G4 foci were observed in nuclei as well as in cytoplasmic areas, including axons, dendrites, and postsynapses. Interestingly, the G4 foci in nuclei show more marked co-localizations with the bright spots of DAPI-positive heterochromatin clusters in mature neurons compared to immature ones. In slices from adult mouse brain, the G4 foci were distributed throughout the brain but were particularly prominent in the hippocampus, olfactory bulb, and cerebellum. In the hippocampus, G4 foci were strongly expressed in neurons and weakly in astrocytes. Consistent with the results in cultured neurons, the nuclear G4 foci were co-localized with heterochromatin in calbindin-positive mature granule cells but less in doublecortin-positive neuronal progenitor cells in the dentate gyrus. Electron microscopic immunolabeling revealed G4 foci on nucleolus-associated chromosomal domains (NADs) and cytoplasm in the adult mouse hippocampal CA1 region. These observations suggest potentially critical roles of G4 in neuronal developmental stages through regulating chromatin structures and cytoplasmic metabolism of RNA.

During development of the peripheral nervous system (PNS), Schwann cells wrap neuronal axons, becoming the myelin sheaths that help axonal functions. While the intercellular signals controlling the myelination process between Schwann cells and peripheral neurons are well studied, the transduction of these signals in Schwann cells still remains elusive. Here, we show that Arf6, an Arf protein of the small GTPase family, is involved in promoting the myelination process. Knockdown of Arf6 with the small-interfering (si)RNA in primary Schwann cells markedly decreases dibutyl-cyclic AMP-induced myelin marker protein expression, indicating that Arf6 plays a role in differentiation-like phenotypic changes. To obtain in vivo evidence, we generated small-hairpin (sh)RNA transgenic mice targeting Arf6 for Schwann cells. Transgenic mice exhibited reduced myelin thickness compared to littermate controls, consistent with the defective myelin formation observed in the transgenic mouse-derived Schwann cell and neuronal culture system. Transgenic mice also exhibited decreased phosphorylation of myelination-related signaling molecules such as Akt kinase cascade proteins as well as downregulation of myelin marker proteins. These results suggest that signaling through Arf6 is required for Schwann cell myelination, adding Arf6 to the list of intracellular signaling molecules involved in the myelination process.

In postnatal development of the peripheral nervous system (PNS), Schwann cells differentiate to insulate neuronal axons with myelin sheaths, increasing the nerve conduction velocity. To produce the mature myelin sheath with its multiple layers, Schwann cells undergo dynamic morphological changes. While extracellular molecules such as growth factors and cell adhesion ligands are known to regulate the myelination process, the intracellular molecular mechanism underlying myelination remains unclear. In this study, we have produced Schwann cell-specific conditional knockout mice for cytohesin-2, a guanine-nucleotide exchange factor (GEF) specifically activating Arf6. Arf6, a member of the Ras-like protein family, participates in various cellular functions including cell morphological changes. Cytohesin-2 knockout mice exhibit decreased Arf6 activity and reduced myelin thickness in the sciatic nerves, with decreased expression levels of myelin protein zero (MPZ), the major myelin marker protein. These results are consistent with those of experiments in which Schwann cell-neuronal cultures were treated with pan-cytohesin inhibitor SecinH3. On the other hand, the numbers of Ki67-positive cells in knockout mice and controls are comparable, indicating that cytohesin-2 does not have a positive effect on cell numbers. Thus, signaling through cytohesin-2 is required for myelination by Schwann cells, and cytohesin-2 is added to the list of molecules known to underlie PNS myelination.

Sirtuins (SIRT1-7) are NAD+-dependent deacetylase/deacylases that regulate a wide variety of biological functions. Although the roles of sirtuins in cartilage homeostasis and cartilage diseases have been well studied, there is no information on the contribution of SIRT7 to cartilage homeostasis and osteoarthritis (OA) pathologies. Here, we demonstrate that Sirt7 knockout mice are resistant to the development of aging-associated OA and forced exercise-induced OA. Attenuation of Sirt7 in the murine chondrogenic cell line ATDC5 increased the deposition of a glycosaminoglycan-rich extracellular matrix and the mRNA expression of extracellular matrix components such as Col2a1 and Acan. Mechanistically, we found that SIRT7 suppressed the transcriptional activity of SOX9, which is an important transcription factor in chondrocytes, and that the enzymatic activity of SIRT7 was required for its function. Our results indicate that SIRT7 is a novel important regulator of cartilage homeostasis and OA development.

In an embryonic developmental stage of the peripheral nervous system (PNS), Schwann cell precursors migrate along neuronal axons to their final destinations. After birth, they eventually wrap around individual axons to form myelin sheaths, which insulate axons to increase the nerve conduction velocity. Some growth factors and adhesion molecules are known to control these developmental stages from in the fish to in the mammal. Neuregulin-1 (NRG1), which is composed of many alternative splicing variants, is such a growth factor. Among these variants, the type III isoform of NRG1, interacting with ErbB2 and ErbB3 receptors on Schwann cells, plays an essential role in myelination in the fish and the mammal. NRG1 type III is also known to promote migration of fish Schwann cell precursors; however, it still remains to be clarified whether mammalian type III isoform does it. We have therefore generated type III isoform-specific knockout mice in inbred strain. The mice result in delayed migration of the precursors from the dorsal to ventral root via a peripheral ganglion, comparing littermate controls. Similar results are observed in an in vitro migration assay using reaggregated Schwann cell precursors. Furthermore, the knockout mice exhibit reduced myelin thickness, consistent with the established role of NRG1 type III in myelination. These results indicate that in mice, NRG1 type III plays a key role not only in myelination but also in migration.

Hepatic steatosis (HS) has a negative effect on liver regeneration, but different pathophysiologies of HS may lead to different outcomes. Male Sprague-Dawley rats were fed a high fructose (66% fructose; H-fruc), high fat (54% fat; H-fat), or control chow diet for 4 weeks. Based on hepatic triglyceride content and oil red O staining, HS developed in the H-fruc group, but was less severe compared to the H-fat group. Hepatic mRNA expression levels of fatty acid synthase and fructokinase were increased and those of carnitine palmitoyltransferase-1 and peroxisome proliferator-activated receptor-α were decreased in the H-fruc group compared to the H-fat group. Liver regeneration after 70% partial hepatectomy (PHx) was evaluated by measuring the increase in postoperative liver mass and PCNA-positive hepatocytes, and was impaired in the H-fruc group compared to the H-fat and control groups on days 3 and 7. Serum levels of tumor necrosis factor-α, interleukin-6 and hepatocyte growth factor did not change significantly after PHx. In contrast, serum TGF-β1 levels were slightly but significantly lower in the control group on day 1 and in the H-fat group on day 3 compared to the level in each group on day 0, and then gradually increased. However, the serum TGF-β1 level did not change after PHx in the H-fruc group. These results indicate that impairment of liver regeneration after PHx in HS is related to the cause, rather than the degree, of steatosis. This difference may result from altered metabolic gene expression profiles and potential dysregulation of TGF-β1 expression.

[Arg(8)]-vasopressin (AVP) plays a crucial role in regulating body fluid retention, which is mediated through the vasopressin V(2) receptor in the kidney. In addition, AVP is involved in the regulation of glucose homeostasis via vasopressin V(1A) and vasopressin V(1B) receptors. Our previous studies demonstrated that vasopressin V(1A) receptor-deficient (V(1A)R-/-) and V(1B) receptor-deficient (V(1B)R-/-) mice exhibited hyperglycemia and hypoglycemia with hypoinsulinemia, respectively. These findings indicate that vasopressin V(1A) receptor deficiency results in decreased insulin sensitivity whereas vasopressin V(1B) receptor deficiency results in increased insulin sensitivity. In addition, vasopressin V(1A) and vasopressin V(1B) receptor double-deficient (V(1AB)R-/-) mice exhibited impaired glucose tolerance, suggesting that the effects of vasopressin V(1B) receptor deficiency do not influence the development of hyperglycemia promoted by vasopressin V(1A) receptor deficiency, and that the blockage of both receptors could lead to impaired glucose tolerance. However, the contributions of the entire AVP/vasopressin receptors system to the regulation of blood glucose have not yet been clarified. In this study, to further understand the role of AVP/vasopressin receptors signaling in blood glucose regulation, we assessed the glucose tolerance of AVP-deficient homozygous Brattleboro (di/di) rats using an oral glucose tolerance test (GTT). Plasma glucose and insulin levels were consistently lower in homozygous di/di rats than in heterozygous di/+ rats during the GTT, suggesting that the blockage of all AVP/vasopressin receptors resulting from the AVP deficiency could lead to enhanced glucose tolerance.

A system for assessing the anti-inflammatory effect of food factors was developed by establishing a co-culture system with intestinal epithelial Caco-2 cells (apical side) and macrophage RAW264.7 cells (basolateral side). In this system, the stimulation of RAW264.7 cells with lipopolysaccharide was followed by a decrease in transepithelial electrical resistance, which is a marker of the integrity of the Caco-2 monolayer and an increase in TNF-alpha production from RAW264.7 cells and IL-8 mRNA expression in Caco-2 cells. Treatment with anti-TNF-alpha antibodies or budesonide suppressed in increase in TNF-alpha production and IL-8 mRNA expression. These results indicated that this co-culture model could imitate the gut inflammation in vivo. In addition, fucoidan, sulphated polysaccharides from brown algae, was employed as a candidate of evolution and added to the apical side of this model. Fucoidan suppressed IL-8 gene expression through a reduction in TNF-alpha production from RAW264.7 cells stimulated with lipopolysaccharide.

Myelin-forming glial cells undergo dynamic morphological changes in order to produce mature myelin sheaths with multiple layers. In the central nervous system (CNS), oligodendrocytes differentiate to insulate neuronal axons with myelin sheaths. Myelin sheaths play a key role in homeostasis of the nervous system, but their related disorders lead not only to dismyelination and repeated demyelination but also to severe neuropathies. Hereditary hypomyelinating leukodystrophies (HLDs) are a group of such diseases affecting oligodendrocytes and are often caused by missense mutations of the respective responsible genes. Despite increasing identification of gene mutations through advanced nucleotide sequencing technology, studies on the relationships between gene mutations and their effects on cellular and subcellular aberrance have not followed at the same rapid pace. In this study, we report that an HLD4-associated (Asp-29-to-Gly) mutant of mitochondrial heat shock 60-kDa protein 1 (HSPD1) causes short-length morphologies and increases the numbers of mitochondria due to their aberrant fission and fusion cycles. In experiments using a fluorescent dye probe, this mutation decreases the mitochondrial membrane potential. Also, mitochondria accumulate in perinuclear regions. HLD4-associated HSPD1 mutant blunts mitochondrial dynamics, probably resulting in oligodendrocyte malfunction. This study constitutes a first finding concerning the relationship between disease-associated HSPD1 mutation and mitochondrial dynamics, which may be similar to the relationship between another disease-associated HSPD1 mutation (MitCHAP-60 disease) and aberrant mitochondrial dynamics.

We have established that differing effects of alpha1-adrenergic receptor (AR) subtypes on cell proliferation are due to differential coupling to the Gs/cAMP pathway; thus, both alpha1A- and alpha1B-ARs couple to Gs, while alpha1D-AR does not. To identify the region responsible for this difference in subtype-specific Gs coupling, we constructed a series of chimeric and a set of point-mutated human alpha1A- and alpha1D-ARs, and examined their signaling ability. Here, we show that the amino acid residues Thr 136 and Val138 in the intracellular loop II of the human alpha1A-AR are intimately involved with Gs coupling.

The myelin sheath insulates neuronal axons and markedly increases the nerve conduction velocity. In the peripheral nervous system (PNS), Schwann cell precursors migrate along embryonic neuronal axons to their final destinations, where they eventually wrap around individual axons to form the myelin sheath after birth. ErbB2 and ErbB3 tyrosine kinase receptors form a heterodimer and are extensively expressed in Schwann lineage cells. ErbB2/3 is thought to be one of the primary regulators controlling the entire Schwann cell development. ErbB3 is the bona fide Schwann cell receptor for the neuronal ligand neuregulin-1. Although ErbB2/3 is well known to regulate both Schwann cell precursor migration and myelination by Schwann cells in fishes, it still remains unclear whether in mammals, ErbB2/3 actually regulates Schwann cell precursor migration. Here, we show that knockdown of ErbB3 using a Schwann cell-specific promoter in mice causes delayed migration of Schwann cell precursors. In contrast, littermate control mice display normal migration. Similar results are seen in an in vitro migration assay using reaggregated Schwann cell precursors. Also, ErbB3 knockdown in mice reduces myelin thickness in sciatic nerves, consistent with the established role of ErbB3 in myelination. Thus, ErbB3 plays a key role in migration, as well as in myelination, in mouse Schwann lineage cells, presenting a genetically conservative role of ErbB3 in Schwann cell precursor migration.

We have examined the effect of inflammatory cytokines on the platelet activation. IL-1 beta and IFN-gamma were found to enhance the adhesion of thrombin-treated platelets to monocytic leukemia cells (U937), when the adhesion was assayed by platelet-mediated cell agglutination. The agglutination was inhibited by a monoclonal anti-GMP140 antibody or EDTA, suggesting that the enhanced platelet adhesion to the leukemic cells was mediated by GMP140. In addition, these cytokines also increased the release of 5-HT from platelets in the presence of a low concentration of thrombin. These data suggest that platelet functions are regulated by the cytokines and that activated platelets participate in inflammatory process.

While a mother's excessive alcohol consumption during pregnancy is known to have adverse effects on fetal neural development, little is known about the underlying mechanism of these effects. In order to investigate these mechanisms, we investigated the toxic effect of ethanol (ETOH) on neural stem/precursor cell (NSC) proliferation. In cultures of NSCs, phospholipase D (PLD) is activated following stimulation with epidermal growth factor (EGF) and fibroblast growth factor 2 (FGF2). Exposure of NSCs to ETOH suppresses cell proliferation, while it has no effect on cell death. Phosphatidic acid (PA), which is a signaling messenger produced by PLD, reverses ETOH inhibition of NSC proliferation. Blocking the PLD signal by 1-butanol suppresses the proliferation. ETOH-induced suppression of NSC proliferation and the protective effect of PA for ETOH-induced suppression are mediated through extracellular signal-regulated kinase signaling. These results indicate that exposure to ETOH impairs NSC proliferation by altering the PLD signaling pathway.

Amino acids regulate cellular functions in a variety of cell types. Most notably, leucine stimulates protein production through the mammalian target of rapamycin (mTOR)-dependent signaling pathway. We investigated the effect of amino acids on hepatocyte growth factor (HGF) production. Treatment with glutamine and proline, as well as leucine, increased HGF levels in the culture medium of a rat hepatic stellate cell clone in a dose-dependent manner. Up-regulation of phosphorylation of 70 kDa ribosomal protein S6 kinase and eukaryotic initiation factor 4E-binding protein 1 was not apparent in the cells after treatment with glutamine or proline. When rats received injections of glutamine or proline, hepatic and circulating HGF levels increased and peaked around 12 h after treatment. Glutamine and proline may have the potential to stimulate HGF production but the mechanism underlying this stimulation seems not to be through the mTOR-dependent signaling pathway.

Branched chain amino acids modulate various cellular functions in addition to providing substrates for the production of proteins. We examined the mechanism underlying the stimulation by leucine of hepatocyte growth factor (HGF) production by hepatic stellate cells. Both p70 S6 kinase activity and phosphorylation of eukaryotic initiation factor 4E-binding protein 1 (4E-BP1) were up-regulated rapidly after leucine treatment of a rat hepatic stellate cell clone. No such activation was observed following treatment with valine or isoleucine. Rapamycin, an inhibitor of mammalian target of rapamycin (mTOR), suppressed leucine-induced activation of p70 S6 kinase and 4E-BP1 and negated the stimulatory effect of leucine on HGF production. An mTOR-dependent signaling pathway mediates the stimulatory effect of leucine on the production of HGF by hepatic stellate cells.

Valproic acid (VPA), which has a wide range of therapeutic applications, is known as a potent teratogen that induces neural tube defects in vertebrates. Here, we have characterized the tissue-specific, embryotoxic effects of VPA on developmental processes using a novel system with differentiating mouse ES cells. Under our cultivating condition, ES cells differentiated into cardiomyocytes, although various cell types can be differentiated. VPA affected cell viability and differentiation from undifferentiated ES cells to cardiomyocytes in a dose-dependent manner. The analysis of tissue-specific markers also revealed that VPA potently inhibited mesodermal and endodermal development but promoted neuronal differentiation in a lineage-specific manner. Taking the in vivo teratogenicity of VPA into account, this assay system could be useful in predicting the degree of embryotoxicity of VPA. We, thus, propose that the in vivo embryotoxic effects of various medicines can be estimated fast and accurately using this in vitro cell differentiation system.

Sphingosine 1-phosphate (S1P) is known to regulate cell proliferation, apoptosis, and motility. Recently, we have reported that S1P and its analogue dihydro-S1P (DHS1P) promote proliferation of rat cultured mesangial cells. To investigate the signaling mechanisms underlying S1P- and DHS1P-induced mesangial cell proliferation, we performed cDNA microarray analysis of gene expression during mesangial cell proliferation. In terms of the overall pattern, gene expression waves induced by S1P and DHS1P were similar to those induced by a potent mesangial mitogen platelet-derived growth factor (PDGF), whereas we found several genes, such as two growth factors, connective tissue growth factor (CTGF) and heparin-binding EGF-like growth factor (HB-EGF), which were induced by the sphingolipids, but not by PDGF. Cluster analysis also identified calcium-dependent molecules highly expressed in DHS1P-stimulated cells compared to S1P-stimulated cells. Calcium mobilization analysis showed that DHS1P had higher magnitudes of intracellular calcium mobilization than S1P, suggesting that S1P and DHS1P differentially regulate intracellular calcium mobilization, possibly leading to different gene expression in mesangial cells. The large-scale monitoring of gene expression performed here allows us to identify S1P-induced transcriptional properties during mesangial cell proliferation.

Hepatocyte growth factor (HGF) is a heterodimer protein, derived from an inactive single-chain precursor (pro-HGF) by the proteolytic cleavage at Arg-Val site. Using a fluorogenic substrate Ac-Lys-Thr-Lys-Gln-Leu-Arg-MCA corresponding to the sequence around the cleavage site of pro-HGF, HGF-converting enzyme was purified from fetal bovine serum. The enzyme is a heterodimer molecule with an apparent molecular weight of about 90-kDa and is composed of a 65-kDa heavy-chain and a 32-kDa light-chain. The enzyme belongs to the serine-protease family and has an optimal pH around 8. The enzyme preferentially cleaved MCA-substrates containing the processing site of pro-HGF. The purified enzyme converted pro-HGF to a two-chain mature form of HGF. The enzyme-treated pro-HGF had mitogenic activity on primary cultured hepatocytes. Thus, the enzyme is likely to be involved in pro-HGF activation in vivo. The enzyme activity in rat serum was 9-fold higher than that in the plasma. This, together with the heterodimeric structure of the enzyme, suggests that the HGF-converting enzyme is activated by another protease in response to a trigger such as blood coagulation or tissue injury.

Rabbit platelets rapidly incorporated methyl groups of [3H] methionine to phosphatidylcholine (PC). Rabbit platelets also incorporated [3H]choline to PC, but the rate of incorporation was far lower than that of [3H]methionine. Further fractionation of labeled PC revealed that a considerable amount of arachidonyl PC was synthesized via the N-methylation pathway. Thrombin stimulation resulted in a release of arachidonic acid from PC, and not from phosphatidylethanolamine (PE). These observations suggest that the N-methylation pathway plays an important role in the intracellular mobilization of arachidonic acid from the PE fraction to the PC fraction, this fraction being more sensitive to the hydrolysis with phospholipase A2 during platelet activation.