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In this report, by using synchrotron radiation X-ray small-angle scattering, we show the structural characteristics of the ganglioside aggregates depending on both the temperature and oligosaccharide chain. The experimental results of the aqueous solutions containing GM1, GD1, or crude mixture gangliosides show that the elevation of temperature induces the change of the micellar structure accompanied by the change of the internal scattering density distribution. These changes are well described by applying a shell-modeling method to the observed scattering curves since the shell-modeling method can determine the intramicellar structures. A noticeable common feature found in the thermotropic transition of the ganglioside micelles is the contraction of the micellar dimension resulting from the shrinkage of the hydrophilic region of the micelle. The shrinkage amounts to 20–30% of the thickness of the hydrophilic region, suggesting the structural change of the oligosaccharide chains of the gangliosides from an extended conformation to a compact one. The differences of the thermotropic stability depending on the number of the sialic acids of the oligosaccharide chain were also observed. The present results which suggest a thermal perturbation in the physiological range 20–50°C induces a conformational change of the oligosaccharide chains of ganglioside molecules very sensitively.

等温加熱による鶏卵白リゾチーム (HEWL) の三次構造の転移およびゲル化のpH・イオン強度依存性について, 時分割放射光X線小角散乱測定による検討を行った. その結果, 85℃, 65分のアニールにより, 0.1M NaCl存在下のpH5.1～7.7, 5%w/v HEWL溶液はゲル化を示し, 三次構造の転移およびゲル化はpHの上昇に伴って促進された. 一方, NaCl非存在下のpH3.0～75, 5%w/v HEWL溶液はゲル化を示さず, また, タンパク質分子の三次構造も完全なアンフォールディングを示さなかった. これは, タンパク質粒子間の静電的反発力が, タンパク質分子の構造安定化と凝集体形成の抑制に寄与していることを示唆する.

Dynamics and phase behavior of biological supermacromolecular (tobacco-mosaic virus) suspensions under magnetic field were investigated using a synchrotron-radiation time-resolved x-ray-scattering method. By increasing the molecular concentration, a nematic-to-smectic phase transition appeared and a transverse correlation within layers became evident at the highest concentration. A phase diagram depending on both magnetic-field strength and molecular concentration was presented. A dependence of the orientational relaxation time on magnetic-field strength in a nematic phase was also measured, which is explained as a magnetically induced collective rotational motion of the constituents within the nematic liquid crystal microdomains with an identical rotational correlation length. The application of magnetic field to a nematic phase suspension enhanced an evident nearest-neighbor correlation in short range which is different from a transverse ordering within layers in a smectic phase, suggesting a mesophase with a partially ordered radial density fluctuations. In contrast, the application of a magnetic field in the smectic phase did not affect the macromolecular arrangement.

∆1-Tetrahydrocannabinolic acid (THCA) synthase catalyzes the oxidative cyclization of cannabigerolic acid (CBGA) into THCA, the precursor of the primary psychoactive agent ∆1-tetrahydrocannabinol in Cannabis sativa . The enzyme was overproduced in insect cells, purified, and crystallized in order to investigate the structure–function relationship of THCA synthase, and the tertiary structure was determined to 2.75 A resolution by X-ray crystallography ( R cryst = 19.9%). The THCA synthase enzyme is a member of the p -cresol methyl-hydroxylase superfamily, and the tertiary structure is divided into two domains (domains I and II), with a flavin adenine dinucleotide coenzyme positioned between each domain and covalently bound to His114 and Cys176 (located in domain I). The catalysis of THCA synthesis involves a hydride transfer from C3 of CBGA to N5 of flavin adenine dinucleotide and the deprotonation of O6′ of CBGA. The ionized residues in the active site of THCA synthase were investigated by mutational analysis and X-ray structure. Mutational analysis indicates that the reaction does not involve the carboxyl group of Glu442 that was identified as the catalytic base in the related berberine bridge enzyme but instead involves the hydroxyl group of Tyr484. Mutations at the active‐site residues His292 and Tyr417 resulted in a decrease in, but not elimination of, the enzymatic activity of THCA synthase, suggesting a key role for these residues in substrate binding and not direct catalysis.

Nucleoside diphosphate kinase (NDK) is known to form homotetramers or homohexamers. To clarify the oligomer state of NDK from moderately halophilic Halomonas sp. 593 (HaNDK), the oligomeric state of HaNDK was characterized by light scattering followed by X-ray crystallography. The molecular weight of HaNDK is 33,660, and the X-ray crystal structure determination to 2.3 and 2.7 Å resolution showed a dimer form which was confirmed in the different space groups of R3 and C2 with an independent packing arrangement. This is the first structural evidence that HaNDK forms a dimeric assembly. Moreover, the inferred molecular mass of a mutant HaNDK (E134A) indicated 62.1-65.3 kDa, and the oligomerization state was investigated by X-ray crystallography to 2.3 and 2.5 Å resolution with space groups of P2(1) and C2. The assembly form of the E134A mutant HaNDK was identified as a Type I tetramer as found in Myxococcus NDK. The structural comparison between the wild-type and E134A mutant HaNDKs suggests that the change from dimer to tetramer is due to the removal of negative charge repulsion caused by the E134 in the wild-type HaNDK. The higher ordered association of proteins usually contributes to an increase in thermal stability and substrate affinity. The change in the assembly form by a minimum mutation may be an effective way for NDK to acquire molecular characteristics suited to various circumstances.

More than 90% of the human genome have been found to be transcribed and most of the transcripts are noncoding (nc) RNAs (Willingham et al., Science 309:1570-1573, 2005; ENCODE-consortium, Science 306:636-640, 2004; Carninci et al., Science 309:1559-1563, 2005; Bertone et al., Science 306:2242-2246, 2004). Studies on ncRNAs have been radically progressed mainly regarding microRNAs, piRNAs, siRNAs, and related small ncRNAs of which length are relatively short nucleotides (Fire et al., Nature 391:806-811, 1998; Filipowicz et al., Nat Rev Genet 9:102-114, 2008; Lau et al., Science 313:363-367, 2006; Brennecke et al., Science 322:1387-1392, 2008; Siomi and Siomi, Nature 457:396-404, 2009). These small RNAs play roles in regulation of translation and gene silencing while long ncRNAs with length more than 200 nucleotides have been emerging and turn out to be involved in regulation of transcription (Kapranov et al., Science 316:1484-1488, 2007; Ponting et al., Cell 136:629-641, 2009; Kurokawa et al., RNA Biol 6:233-236, 2009). Recently, we have identified novel, long ncRNAs bearing capability of repression of transcription (Wang et al., Nature 454:126-130, 2008).RNA-binding protein, translocated in liposarcoma (TLS), binds CREB-binding protein CBP/adenovirus p300 and inhibits their histone acetyltransferase (HAT) activities (Wang et al., Nature 454:126-130, 2008). The HAT inhibitory activity of TLS requires specific binding of RNA. The systematic evolution of ligands by exponential enrichment experiments with randomized sequences revealed that TLS specifically recognizes RNA oligonucleotides containing GGUG as a consensus sequence although the GGUG sequence is not an absolute requirement for the TLS binding (Lerga et al., J Biol Chem 276:6807-6816, 2001). TLS is specifically recruited to the CBP/p300-associated binding sites of the cyclin D1 gene (CCND1) and the cyclin E1 gene (CCNE1) promoters (Wang et al., Nature 454:126-130, 2008; Impey et al., Cell 119:1041-1054, 2004). Our extensive exploration for naturally occurring RNA molecule that binds TLS has indicated that long ncRNAs (promoter-associated ncRNAs: pncRNAs) transcribed from the CCND1 promoter bind TLS and inhibit the HAT activities on the sites to repress the transcription of the CCND1 gene (Wang et al., Nature 454:126-130, 2008). We have optimized RT-PCR, chromatin immunoprecipitation, RNA immunoprecipitation, and RNA gel-shift assay in order to detect these pncRNAs. The methods that we have developed successfully identified these low-abundant, long ncRNAs and provide the data showing that the CCND1 pncRNAs bind TLS and induce its HAT inhibitory activity to repress the transcription of CCND1 gene upon genotoxic stress.

More than 90% of the human genome have been found to be transcribed and most of the transcripts are noncoding (nc) RNAs (Willingham et al., Science 309:1570-1573, 2005; ENCODE-consortium, Science 306:636-640, 2004; Carninci et al., Science 309:1559-1563, 2005; Bertone et al., Science 306:2242-2246, 2004). Studies on ncRNAs have been radically progressed mainly regarding microRNAs, piRNAs, siRNAs, and related small ncRNAs of which length are relatively short nucleotides (Fire et al., Nature 391:806-811, 1998; Filipowicz et al., Nat Rev Genet 9:102-114, 2008; Lau et al., Science 313:363-367, 2006; Brennecke et al., Science 322:1387-1392, 2008; Siomi and Siomi, Nature 457:396-404, 2009). These small RNAs play roles in regulation of translation and gene silencing while long ncRNAs with length more than 200 nucleotides have been emerging and turn out to be involved in regulation of transcription (Kapranov et al., Science 316:1484-1488, 2007; Ponting et al., Cell 136:629-641, 2009; Kurokawa et al., RNA Biol 6:233-236, 2009). Recently, we have identified novel, long ncRNAs bearing capability of repression of transcription (Wang et al., Nature 454:126-130, 2008).RNA-binding protein, translocated in liposarcoma (TLS), binds CREB-binding protein CBP/adenovirus p300 and inhibits their histone acetyltransferase (HAT) activities (Wang et al., Nature 454:126-130, 2008). The HAT inhibitory activity of TLS requires specific binding of RNA. The systematic evolution of ligands by exponential enrichment experiments with randomized sequences revealed that TLS specifically recognizes RNA oligonucleotides containing GGUG as a consensus sequence although the GGUG sequence is not an absolute requirement for the TLS binding (Lerga et al., J Biol Chem 276:6807-6816, 2001). TLS is specifically recruited to the CBP/p300-associated binding sites of the cyclin D1 gene (CCND1) and the cyclin E1 gene (CCNE1) promoters (Wang et al., Nature 454:126-130, 2008; Impey et al., Cell 119:1041-1054, 2004). Our extensive exploration for naturally occurring RNA molecule that binds TLS has indicated that long ncRNAs (promoter-associated ncRNAs: pncRNAs) transcribed from the CCND1 promoter bind TLS and inhibit the HAT activities on the sites to repress the transcription of the CCND1 gene (Wang et al., Nature 454:126-130, 2008). We have optimized RT-PCR, chromatin immunoprecipitation, RNA immunoprecipitation, and RNA gel-shift assay in order to detect these pncRNAs. The methods that we have developed successfully identified these low-abundant, long ncRNAs and provide the data showing that the CCND1 pncRNAs bind TLS and induce its HAT inhibitory activity to repress the transcription of CCND1 gene upon genotoxic stress.

The G-quadruplex nucleic acid structural motif is a target for designing molecules with potential anticancer properties. To achieve therapeutic selectivity by targeting the G-quadruplex, the molecules must be able to differentiate between the DNA of different G-quadruplexes. We recently reported that the Arg-Gly-Gly repeat (RGG) of the C-terminus in Ewing's sarcoma protein (EWS), which is a group of dominant oncogenes that arise due to chromosomal translocations, is capable of binding to G-quadruplex telomere DNA and RNA via arginine residues and stabilize the G-quadruplex DNA form in vitro. Here, we show that the RGG of EWS binds preferentially to G-quadruplexes with longer loops, which is not related to the topology of the G-quadruplex structure. Moreover, the G-quadruplex DNA binding of the RGG in EWS depends on the phosphate backbone of the loops in the G-quadruplex DNA. We also investigated the G-quadruplex DNA binding activity of the N- and C-terminally truncated RGG to assess the role of the regions in the RGG in G-quadruplex DNA binding. Our findings indicate that the RGG and the other arginine-rich motif of residues 617-656 of the RGG in EWS are important for the specific binding to G-quadruplex DNA. These findings will contribute to the development of molecules that selectively target different G-quadruplex DNA.

The Ewing's sarcoma (EWS) oncogene contains an N-terminal transcription activation domain and a C-terminal RNA-binding domain. Although the EWS activation domain is a potent transactivation domain that is required for the oncogenic activity of several EWS fusion proteins, the normal role of intact EWS is poorly characterized because little is known about its nucleic acid recognition specificity. Here we show that the Arg-Gly-Gly (RGG) domain of the C-terminal in EWS binds to the G-rich single-stranded DNA and RNA fold in the G-quadruplex structure. Furthermore, inhibition of DNA polymerase on a template containing a human telomere sequence in the presence of RGG occurs in an RGG concentration-dependent manner by the formation of a stabilized G-quadruplex DNA-RGG complex. In addition, mutated RGG containing Lys residues replacing Arg residues at specific Arg-Gly-Gly sites and RGG containing Arg methylated by protein arginine N-methyltransferase 3 decrease the binding ability of EWS to G-quadruplex DNA and RNA. These findings suggest that the RGG of EWS binds to G-quadruplex DNA and RNA via the Arg residues in it.

Crystallization of a large single crystal of a B-DNA decamer, d(CCATTAATGG), for a neutron-diffraction experiment has been accomplished by an analysis of its solubility phase diagram and a large single crystal was successfully crystallized at around the minimum solubility point of the oligonucleotide: 30%(v/v) MPD, 100 mM MgCl(2) pD 6.6 using 0.4 ml D(2)O solutions of the DNA (sample concentration 1.5 mM). It is confirmed that the resulting crystal (dimensions: 1.7 x 1.3 x 0.6 mm) diffracts sufficiently well for neutron data collection.

HIV-1 protease is a dimeric aspartic protease that plays an essential role in viral replication. To further understand the catalytic mechanism and inhibitor recognition of HIV-1 protease, we need to determine the locations of key hydrogen atoms in the catalytic aspartates Asp-25 and Asp-125. The structure of HIV-1 protease in complex with transition-state analog KNI-272 was determined by combined neutron crystallography at 1.9-Å resolution and X-ray crystallography at 1.4-Å resolution. The resulting structural data show that the catalytic residue Asp-25 is protonated and that Asp-125 (the catalytic residue from the corresponding diad-related molecule) is deprotonated. The proton on Asp-25 makes a hydrogen bond with the carbonyl group of the allophenylnorstatine (Apns) group in KNI-272. The deprotonated Asp-125 bonds to the hydroxyl proton of Apns. The results provide direct experimental evidence for proposed aspects of the catalytic mechanism of HIV-1 protease and can therefore contribute substantially to the development of specific inhibitors for therapeutic application.

By using solvent contrast variation method of small-angle neutron scattering, we have studied the thermal structural transition of glycosphingolipid micelle (0.5% w/v monosialogangliosides in 50 mM Hepes buffer at pH 7). The present results clearly indicate that the thermal structural transition accompanies a change of the intermicellar scattering density distribution, which results from the dehydration of the polar head (oligosaccharide chain with acidic sugar) region of the micelle. The present experimental evidences strongly support our previous results obtained by using small-angle X-ray scattering and shell-modeling methods.

By using both wide-angle neutron and small-angle X-ray scattering techniques, we have studied the denaturation processes of hen egg-white lysozyme (HEWL) and bovine α-lactalbumin (BLA) in dimethyl sulfoxide (DMSO)/water mixtures. Although these proteins are known to be highly homologous, the present results indicate that the structure of BLA is liable to be destroyed by the addition of DMSO in comparison with the case of HEWL. DMSO-induced denaturation is suggested to relate to the collapse of hydration shell surrounding the protein surface.

By using neutron spin echo and synchrotron radiation small-angle X-ray scattering, we have studied the structure and dynamics of water/sodium bis(2-ethylhexyl)sulfosuccinate/d 16 - n -heptane microemulsion with varying water/surfactant molar ratio, w 0 . We have found the anomalous q -dependence of the effective diffusion coefficient D eff at low w 0 whose peak-position at low w 0 does not satisfy the predicted relation of qR = π ( R , microemulsion radius). This would relate to the presence of the oligomeric phase at low w 0 observed by SAXS.

In this paper we treat the interaction of acidic-glycolipids with proteins. We used monosialoganglioside, disialoganglioside, and ganglioside mixture. The proteins used were albumins whose surfaces are modified differently with mono- and disaccharides. By using X-ray and neutron-scattering techniques we have found that the interactions of mono- and disialogangliosides with albumins having monosaccharides occur less destructively for ganglioside aggregate structures at low protein concentrations and that the interaction of monosialogangliosides with albumins having disaccharides occurs very destructively, suggesting a strong interaction between them compared with other cases.

Recently, we showed that the hydrolysis of some esters catalyzed by α-chymotrypsin entrapped in sodium bis(2-ethylhexyl)sulfosuccinate (AOT) microemulsions is enhanced at low water-surfactant molar ratio w 0 ( = [H 2 O]/[AOT]) and that the internal interface structure and the oligomeric phase of the AOT microemulsions below this w 0 range would play an important role for the acceleration of the metabolic turnover. To clarify the relation between the appearance of superactivity and the internal structures of the AOT microemulsions, we have carried out small-angle neutron-scattering experiments. In this report, we treat the AOT/water/hexane or heptane system by using solvent-contrast variation method, and we discuss a change of the excluded volume of AOT molecule at low w 0 value.

Many studies of folding-unfolding transition of protein have been carried out for the clarification of the fundamental biological problem of protein folding, namely, how a polypeptide chain attains its native conformation. Although one of the approaches to solve protein-folding mechanisms is evidently to follow denaturation processes of protein, there is little direct evidence showing intramolecular structural changes under thermal perturbation. By using a wide-angle neutron-scattering spectrometer, we have monitored the thermal denaturation process of hen egg-white lysozyme at different pH under the unaggregative condition. The present results show that the temperature elevation gradually induces an intramolecular structural fluctuation prior to a main transition of the tertiary structure without accompanying significant heat absorption, and the multiplicity in the thermal transition observed suggests the presence of hierarchic stability depending on the structural hierarchy which would characterize an energetic landscape of folding-unfolding transition of protein.

In order to reveal the hydration structure of Z-DNA, a neutron diffraction study has been carried out at 1.8 A resolution on a Z-DNA hexamer d(CGCGCG). Neutron diffraction data were collected with the BIX-3 single-crystal diffractometer at the JRR-3 reactor in the Japan Atomic Energy Research Institute (JAERI) using a large crystal (1.6 mm3) obtained from D2O solution. It has been found that almost all the guanine bases have participated in H/D exchange at the C8—H8 group, consistent with the acidic nature of this bond. 44 water molecules were found in the nuclear density maps, of which 29 showed the entire contour of all three atoms (D—O—D). The remaining 15 water molecules had a simple spherical shape, indicating that they were rotationally disordered. An interesting relationship was found between the orientational disorder of the water molecules and their locations. Almost all water molecules in the minor groove were well ordered in the crystal, while 40% of the water molecules in the major groove were rotationally disordered. The hydrogen-bonding networks in the hydration shells have two structural aspects: flexibility and regularity.

Dissimilatory sulfite reductase D (DsrD) from Desulfovibrio vulgaris has been crystallized for a neutron diffraction study. The initial crystals obtained were too small for the neutron experiment. In order to obtain a larger crystal (1 mm3), a combination of two techniques was developed to determine the optimum crystallization conditions: a crystallization phase diagram was obtained, followed by crystal-quality assessment via X-ray diffraction. Using conditions determined in this manner, a large single crystal (1.7 mm3) of DsrD protein was subsequently grown in D(2)O solution by the macroseeding technique. A neutron diffraction experiment was carried out using the BIX-3 diffractometer at the Japan Atomic Energy Research Institute (JAERI), collecting data to 2.4 A resolution from an optimized crystal.

The factors SRC-1, TIF2 and ACTR were identified as interacting with nuclear receptors in a highly ligand-dependent manner. Because the molecular mass of each of these factors is approximately 160 kDa, they are collectively termed p160 coactivators. So far, p160 coactivators have been cloned from human, mouse and Xenopus. We report here the cloning of the chicken homologues of p160 coactivator members. As in human and mouse, chicken has three p160 coactivators. Each gene encodes an approximately 160 kDa protein which exhibits 70-80% amino acid sequence identity to human and mouse p160 coactivators. Chicken p160 coactivators also have the property of interacting with several liganded nuclear receptors. Moreover, we describe an imperfect LXXLL sequence, termed NR box 4, which is located downstream of NR box 3 and conserved between evolutionarily diverse species. The loss of NR box 4 results in a decrease of interaction with the nuclear receptor, which indicates that NR box 4 is required for efficient interaction.

TLS (Translocated in liposarcoma) has been characterized as a rearranged gene in chromosomal translocations specific of human myxoid liposarcoma. The various cellular functions of TLS participating either in transcription or splicing processes are thought the involvement of an interaction of TLS with DNA and/or RNA. To investigate insight into DNA-TLS interaction, we performed Electrophoretic mobility shift assay of TLS with G-quadruplex DNA. It revealed that TLS especially bound to single stranded human telomeric DNA in the presence of potassium ion while it was not able to bind double stranded human telomeric DNA and single stranded human telomeric DNA in the presence of sodium ion.

The TET-family proteins (TAF15, EWS and TLS) are the RNA binding proteins involved in multiple levels of cellular functions. The RNA binding domain of those proteins is known as the important region for cellular functions. But little is known about the RNA binding specificity of TET-family proteins. In order to investigate the RNA binding properties of the TET-family proteins, we performed electrophoretic mobility shift assay using recombinant Flag-tagged TLS and guanine-rich and RNAs. It was found that TLS binds to human telomeric RNA in the presence of KCl, but not in the presence of LiCl.

1 alpha,25-Dihydroxyvitamin D3 [1,25-(OH)2D3], the active form of vitamin D3, has been thought to be a multifunctional agent. In order to discover novel roles of 1,25-(OH)2D3, we have been looking for new genes that are regulated by 1,25-(OH)2D3. Because the actions of 1,25-(OH)2D3 are mediated through the vitamin D receptor (VDR), that is a DNA binding transcription factor, vitamin D regulated genes should have VDR binding sites in their regulatory regions. In this paper, we describe a novel vitamin D response element (VDRE)-containing sequence, clone 3, which was isolated through binding to VDR. DNA sequence analysis of clone 3 did not reveal any significant similarity with sequences reported previously. Clone 3 had two regions consisting of a direct repeated sequence of AGTTCA motifs, both of which bound to VDR independently. Whereas each direct repeat sequence alone could not mediate transcriptional activation efficiently, with their co-existence there was a strong response to 1,25-(OH)2D3, indicating that these two direct repeated sequences act cooperatively.

Chromosomal translocation product, TLS-CHOP (Translocated in liposarcoma-CCAAT/enhancer binding protein homologous protein, also named as FUS-DDIT3), has been thought to be a primary cause of myxoid liposarcoma, but the precise molecular function of TLS-CHOP for oncogenesis still remains to be elucidated. Previously we demonstrated that TLS/FUS interacts with protein arginine methyltransferase 1 (PRMT1), and carboxyl-terminal region of TLS is dimethylated by PRMT1. However, it has been uncovered whether TLS-CHOP function is regulated by PRMT1, and is methylated. Here we indicate that TLS-CHOP is not associated with PRMT1 and less methylated even though TLS-CHOP still possesses several potential arginine methylation sites of TLS. Moreover, we established a stable cell line expressing TLS-CHOP as a model system for studying the molecular function of TLS-CHOP. The TLS-CHOP expressing 293T cells exhibited slight growth retardation and decreased level of integrin 51 protein, a fibronectin receptor. It would be possible that the expression of oncoprotein TLS-CHOP might dysregulate arginine-specific methylation elicited via PRMT1 interacting with methylated TLS.

The vitamin D receptor (VDR) binds to the vitamin D response element (VDRE) in the promoter region of target genes and acts as a ligand-dependent transcriptional regulator. In order to identify novel VDREs and new genes that are regulated by the active form of vitamin D [1,25-(OH)2D3], rat genomic DNA fragments bound by VDR were isolated. One of these fragments, designated as VBF5, mediated 1,25-(OH)2D3-dependent negative regulation. Moreover, a genomic DNA region around VBF5 was transcribed and the transcript was down-regulated by 1,25-(OH)2D3. These data strongly indicate that VBF5 may contain a VDRE regulating negatively an unidentified gene expression.

Excessive accumulation of bone marrow adipocytes observed in senile osteoporosis or age-related osteopenia is caused by the unbalanced differentiation of MSCs into bone marrow adipocytes or osteoblasts. Several transcription factors are known to regulate the balance between adipocyte and osteoblast differentiation. However, the molecular mechanisms that regulate the balance between adipocyte and osteoblast differentiation in the bone marrow have yet to be elucidated. To identify candidate genes associated with senile osteoporosis, we performed genome-wide expression analyses of differentiating osteoblasts and adipocytes. Among transcription factors that were enriched in the early phase of differentiation, Id4 was identified as a key molecule affecting the differentiation of both cell types. Experiments using bone marrow-derived stromal cell line ST2 and Id4-deficient mice showed that lack of Id4 drastically reduces osteoblast differentiation and drives differentiation toward adipocytes. On the other hand knockdown of Id4 in adipogenic-induced ST2 cells increased the expression of Pparγ2, a master regulator of adipocyte differentiation. Similar results were observed in bone marrow cells of femur and tibia of Id4-deficient mice. However the effect of Id4 on Pparγ2 and adipocyte differentiation is unlikely to be of direct nature. The mechanism of Id4 promoting osteoblast differentiation is associated with the Id4-mediated release of Hes1 from Hes1-Hey2 complexes. Hes1 increases the stability and transcriptional activity of Runx2, a key molecule of osteoblast differentiation, which results in an enhanced osteoblast-specific gene expression. The new role of Id4 in promoting osteoblast differentiation renders it a target for preventing the onset of senile osteoporosis., Author Summary Increased bone marrow adiposity is observed in the bone marrow of senile osteoporosis patients. This is caused by unbalanced differentiation of mesenchymal stem cells (MSCs) into osteoblast or adipocyte. Previous reports have indicated that several transcription factors play important roles in determining the direction of MSCs differentiation into osteoblast or adipocyte. So far, little is known about the overall dynamics and regulation of transcription factor expression changes leading to the imbalance of osteoblast and adipocyte differentiation inside the bone marrow. We have performed genome-wide gene expression analyses during the differentiation of MSCs into osteoblast or adipocyte. We identified basic helix-loop-helix transcription factor family member Id4 as a leading candidate controlling the differentiation toward adipocyte or osteoblast. Suppression of Id4 expression in MSCs repressed osteoblast differentiation and increased adipocyte differentiation. In contrast, overexpression of Id4 in MSCs promoted osteoblast differentiation and attenuated adipocyte differentiation. Moreover, Id4-mutant mice showed abnormal accumulation of lipid droplets in bone marrow and impaired bone formation activity. In summary, we have demonstrated a molecular function of Id4 in osteoblast differentiation. The findings revealed that Id4 is a molecular switch enhancing osteoblast differentiation at the expense of adipocyte differentiation.

With the development and use of electromagnetic pumps in metallurgical and atomic industries, the investigation of the flow of electrically conducting fluids in channels under an applied magnetic field is growing in importance. Here is reported an experimental study of transition and turbulent flows in curved channels with rectangular cross section under a transverse magnetic field. Mercury is run in curved channels with relatively small radii of curvature where the secondary flow exerts a strong influence. Several phenomena are reported, which can not be encountered in flows in curved channels of non-magnetic case and MHD flows in straight channels. Besides, the influence of the Reynolds number, the Hartmann number, the channel radius of curvature and the aspect ratio on the resistance coefficient of transition and turbulent flows in curved channels is made clear.

With the development and use of such MHD devices as electromagnetic pumps in metallurgical and atomic industries, and MHD generators, the investigation of the flow of electrically conducting fluids in channels under an applied magnetic field is growing in importance. Here are reported experimental results of laminar and transition flows in a transversely applied magnetic field. Mercury is run in two types of rectangular channels, one having insulating walls, the other with insulating top and bottom walls and conducting side walls. The influence of Reynolds number, the electrical property of the side walls and the aspect rations of channels on such flows in the range of relatively low Hartmann numbers is described.

The effect of a transverse magnetic field on a steady flow of liquid metals in curved rectangular channels is studied theoretically in this paper. A solution is obtained using the boundary layer approximation. The results for the resistance coefficient agree well with the experimental results over wide ranges of Hartmann numbers, Dean numbers and aspect ratios. Besides it is made clear that the suppression effect on the secondary flow by the Lorentz force gets larger with an increasing Hartmann number, so that the value of the resistance coefficient for a flow in a curved channel coincides with that in a straight channel even at high Dean number regime.