Okazaki National Research Institutes

We present possible observing scenarios for the Advanced LIGO, Advanced Virgo and KAGRA gravitational-wave detectors over the next decade, with the intention of providing information to the astronomy community to facilitate planning for multi-messenger astronomy with gravitational waves. We estimate the sensitivity of the network to transient gravitational-wave signals, and study the capability of the network to determine the sky location of the source. We report our findings for gravitational-wave transients, with particular focus on gravitational-wave signals from the inspiral of binary neutron star systems, which are the most promising targets for multi-messenger astronomy. The ability to localize the sources of the detected signals depends on the geographical distribution of the detectors and their relative sensitivity, and [Formula: see text] credible regions can be as large as thousands of square degrees when only two sensitive detectors are operational. Determining the sky position of a significant fraction of detected signals to areas of 5-[Formula: see text] requires at least three detectors of sensitivity within a factor of [Formula: see text] of each other and with a broad frequency bandwidth. When all detectors, including KAGRA and the third LIGO detector in India, reach design sensitivity, a significant fraction of gravitational-wave signals will be localized to a few square degrees by gravitational-wave observations alone.

BACKGROUND: Ror2 is an orphan receptor, belonging to the Ror family of receptor tyrosine kinases. Although Ror2 has been shown to play crucial roles in developmental morphogenesis, the precise signalling events that Ror2 mediates remain elusive. Since Ror2 possesses an extracellular cysteine-rich domain (CRD) that resembles the Wnt-binding sites of the Frizzled (Fz) proteins, it is conceivable that Ror2 interacts with members of the Wnt family. RESULTS: Both Ror2-/- and Wnt5a-/- mice exhibit dwarfism, facial abnormalities, short limbs and tails, dysplasia of lungs and genitals, and ventricular septal defects. In vitro binding assay revealed that Wnt5a binds to the CRD of Ror2. Furthermore, Ror2 associates via its CRD with rFz2, a putative receptor for Wnt5a. Interestingly, Wnt5a and Ror2 activate the non-canonical Wnt pathway, as assessed by activation of JNK in cultured cells and inhibition of convergent extension movements in Xenopus. CONCLUSIONS: Our findings indicate that Wnt5a and Ror2 interact physically and functionally. Ror2 may thus act as a receptor for Wnt5a to activate non-canonical Wnt signalling.

Fibroblast growth factor (FGF) signaling is involved in skeletal development of the vertebrate. Gain-of-function mutations of FGF receptors (FGFR) cause craniosynostosis, premature fusion of the skull, and dwarfism syndromes. Disruption of Fgfr3 results in prolonged growth of long bones and vertebrae. However, the role that FGFs actually play in skeletal development in the embryo remains unclear. Here we show that Fgf18 is expressed in and required for osteogenesis and chondrogenesis in the mouse embryo. Fgf18 is expressed in both osteogenic mesenchymal cells and differentiating osteoblasts during calvarial bone development. In addition, Fgf18 is expressed in the perichondrium and joints of developing long bones. In calvarial bone development of Fgf18-deficient mice generated by gene targeting, the progress of suture closure is delayed. Furthermore, proliferation of calvarial osteogenic mesenchymal cells is decreased, and terminal differentiation to calvarial osteoblasts is specifically delayed. Delay of osteogenic differentiation is also observed in the developing long bones of this mutant. Conversely, chondrocyte proliferation and the number of differentiated chondrocytes are increased. Therefore, FGF18 appears to regulate cell proliferation and differentiation positively in osteogenesis and negatively in chondrogenesis.

Large numbers of apoptotic early diplotene oocytes were observed during the transition from ovary-like undifferentiated gonadal tissue to testes during sex differentiation in presumptive males of the zebrafish (Danio rerio). The percentage of terminal-deoxynucleotidyl-transferase-mediated dUTP nick-end labelling (TUNEL)-positive apoptotic oocytes in the gonads of presumptive males was approximately eight- to 12-fold higher than in genetic all-females. By 29 days post-hatching, all oocytes had disappeared from the gonads of presumptive males. In these males, we also observed apoptotic somatic cells in the ovarian cavity between 23 and 35 days post-hatching. Therefore, the disappearance of oocytes and the decomposition of the ovarian cavity caused by apoptosis during sex differentiation were male-specific events. In genetic all-females, apoptosis in a proportion of early diplotene oocytes was found in the undifferentiated gonads at 15-19 days post-hatching, probably as a result of programmed oocyte loss during ovarian development. These findings suggest that oocyte apoptosis is the mechanism of testicular and ovarian differentiation in zebrafish.

The glutamate transporter GLAST is localized on the cell membrane of mature astrocytes and is also expressed in the ventricular zone of developing brains. To characterize and follow the GLAST-expressing cells during development, we examined the mouse spinal cord by in situ hybridization and immunohistochemistry. At embryonic day (E) 11 and E13, cells expressing GLAST mRNA were present only in the ventricular zone, where GLAST immunoreactivity was associated with most of the cell bodies of neuroepithelial cells. In addition, GLAST immunoreactivity was detected in radial processes running through the mantle and marginal zones. From this characteristic cytology, GLAST-expressing cells at early stages were judged to be radial glia cells. At E15, cells expressing GLAST mRNA first appeared in the mantle zone, and GLAST-immunopositive punctate or reticular protrusions were formed along the radial processes. From E18 to postnatal day (P) 7, GLAST mRNA or its immunoreactivity gradually decreased from the ventricular zone and disappeared from radial processes, whereas cells with GLAST mRNA spread all over the mantle zone and GLAST-immunopositive punctate/reticular protrusions predominated in the neuropils. At P7, GLAST-expressing cells were immunopositive for glial fibrillary acidic protein, an intermediate filament specific to astrocytes. Therefore, the glutamate transporter GLAST is expressed from radial glia through astrocytes during spinal cord development. Furthermore, the distinct changes in the cell position and morphology suggest that both the migration and transformation of radial glia cells begin in the spinal cord between E13 and E15, when the active stage of neuronal migration is over.

Epilepsy is a devastating and poorly understood disease. Mutations in a secreted neuronal protein, leucine-rich glioma inactivated 1 (LGI1), were reported in patients with an inherited form of human epilepsy, autosomal dominant partial epilepsy with auditory features (ADPEAF). Here, we report an essential role of LGI1 as an antiepileptogenic ligand. We find that loss of LGI1 in mice (LGI1(-/-)) causes lethal epilepsy, which is specifically rescued by the neuronal expression of LGI1 transgene, but not LGI3. Moreover, heterozygous mice for the LGI1 mutation (LGI1(+/-)) show lowered seizure thresholds. Extracellularly secreted LGI1 links two epilepsy-related receptors, ADAM22 and ADAM23, in the brain and organizes a transsynaptic protein complex that includes presynaptic potassium channels and postsynaptic AMPA receptor scaffolds. A lack of LGI1 disrupts this synaptic protein connection and selectively reduces AMPA receptor-mediated synaptic transmission in the hippocampus. Thus, LGI1 may serve as a major determinant of brain excitation, and the LGI1 gene-targeted mouse provides a good model for human epilepsy.

Embryonic patterning in Drosophila is regulated by maternal factors. Many such factors become localized as mRNAs within the oocyte during oogenesis and are translated in a spatio-temporally regulated manner. These processes are controlled by trans-acting proteins, which bind to the target RNAs to form a ribonucleoprotein (RNP) complex. We report that a DEAD-box protein, Me31B, forms a cytoplasmic RNP complex with oocyte-localizing RNAs and Exuperantia, a protein involved in RNA localization. During early oogenesis, loss of Me31B causes premature translation of oocyte-localizing RNAs within nurse cells, without affecting their transport to the oocyte. These results suggest that Me31B mediates translational silencing of RNAs during their transport to the oocyte. Our data provide evidence that RNA transport and translational control are linked through the assembly of RNP complex.

The cerebellar inositol 1,4,5-trisphosphate (InsP3) receptor is a high molecular weight glycoprotein abundantly expressed in Purkinje cells. The subunit structure of the InsP3 receptor protein was examined by cross-linking experiments. Agarose-polyacrylamide gel electrophoresis of the cross-linked materials demonstrated that the cerebellar InsPs receptor protein is composed of four noncovalently bound identical subunits each with a M, of 320,000 in both purified and microsome-bound states. Chromatography of the purified receptor on a calmodulin-Sepharose column demonstrated a Ca2+-dependent interaction of the InsP3 receptor with calmodulin. Photoaffinity labeling of the cerebellar microsomal fraction with [ ~t -~~P ] 8azidoadenosine 5'-triphosphate revealed the presence of ATP-binding site in the InsP3 receptor. Scatchard analysis of the purified InsP3 receptor revealed the B,,, and K , values for ATP binding of 2.3 pmol/pg and 17 p ~, respectively. Reconstitution of the purified InsPs receptor into the planar lipid bilayer indicated channel activity in the purified receptor. It exhibited a calcium conductance (26 pS in 53 m M Ca2+) and sodium conductance (21 pS in 100-500 m M asymmetric Na+ solutions) with permeability ratios of P ~J P T ~~~ = 6.3 and P N J P ~, = 5.4. The purified channel was activated with submillimolar ATP in the presence of InsP3 and modified to reach a large conductance state.

Recent molecular genetic studies suggest that the expression of transcription factors in the developing spinal cord helps determine the morphological and physiological properties of neurons. Using the zebrafish preparation, we have examined the properties of neurons marked by alx , a zebrafish homolog of mammalian Chx10 . We performed morphological and physiological studies using transgenic zebrafish expressing fluorescent reporter constructs in cells that had at any time point expressed alx ( alx neurons). Our data reveal that zebrafish alx neurons are all ipsilateral descending neurons that are positive for vesicular glutamate transporter 2, suggesting that they are glutamatergic excitatory interneurons. Patch recordings show that earlier-born neurons are active during stronger movements such as escapes and fast swimming (strong movement class), whereas later-born ones are involved in sustained weak swimming (weak movement class). Paired recordings between alx neurons and motoneurons show that neurons of the strong movement class make frequent monosynaptic excitatory connections onto motoneurons. Thus, neurons of this class are likely premotor interneurons that regulate motoneuron activity during escapes and fast swimming. We also show the existence of a monosynaptic connection between an alx neuron of the weak movement class and a motoneuron. Collectively, our data demonstrate that alx marks ipsilateral descending neurons that are involved in the regulation of motoneuron activity during forms of locomotion, such as escape and swimming.

In the developing spinal cord, signals from the roof plate are required for the development of three classes of dorsal interneuron: D1, D2, and D3, listed from dorsal to ventral. Here, we demonstrate that absence of Wnt1 and Wnt3a, normally expressed in the roof plate, leads to diminished development of D1 and D2 neurons and a compensatory increase in D3 neuron populations. This occurs without significantly altered expression of BMP and related genes in the roof plate. Moreover, Wnt3a protein induces expression of D1 and D2 markers in the isolated medial region of the chick neural plate, and Noggin does not interfere with this induction. Thus, Wnt signaling plays a critical role in the specification of cell types for dorsal interneurons.

The formation of vibrationally excited heme upon photodissociation of carbonmonoxy myoglobin and its subsequent vibrational energy relaxation was monitored by picosecond anti-Stokes resonance Raman spectroscopy. The anti-Stokes intensity of the nu4 band showed immediate generation of vibrationally excited hemes and biphasic decay of the excited populations. The best fit to double exponentials gave time constants of 1.9 +/- 0.6 and 16 +/- 9 picoseconds for vibrational population decay and 3.0 +/- 1.0 and 25 +/- 14 picoseconds for temperature relaxation of the photolyzed heme when a Boltzmann distribution was assumed. The decay of the nu4 anti-Stokes intensity was accompanied by narrowing and frequency upshift of the Stokes counterpart. This direct monitoring of the cooling dynamics of the heme cofactor within the globin matrix allows the characterization of the vibrational energy flow through the protein moiety and to the water bath.

Transient receptor potential V3 (TRPV3) and TRPV4 are heat-activated cation channels expressed in keratinocytes. It has been proposed that heat-activation of TRPV3 and/or TRPV4 in the skin may release diffusible molecules which would then activate termini of neighboring dorsal root ganglion (DRG) neurons. Here we show that adenosine triphosphate (ATP) is such a candidate molecule released from keratinocytes upon heating in the co-culture systems. Using TRPV1-deficient DRG neurons, we found that increase in cytosolic Ca(2+)-concentration in DRG neurons upon heating was observed only when neurons were co-cultured with keratinocytes, and this increase was blocked by P2 purinoreceptor antagonists, PPADS and suramin. In a co-culture of keratinocytes with HEK293 cells (transfected with P2X(2) cDNA to serve as a bio-sensor), we observed that heat-activated keratinocytes secretes ATP, and that ATP release is compromised in keratinocytes from TRPV3-deficient mice. This study provides evidence that ATP is a messenger molecule for mainly TRPV3-mediated thermotransduction in skin.

Myelinated axons are divided into four distinct regions: the node of Ranvier, paranode, juxtaparanode, and internode, each of which is characterized by a specific set of axonal proteins. Voltage-gated Na+ channels are clustered at high densities at the nodes, whereas shaker-type K+ channels are concentrated at juxtaparanodal regions. These channels are separated by the paranodal regions, where septate-like junctions are formed between the axon and the myelinating glial cells. Although oligodendrocytes and myelin sheaths are believed to play an instructive role in the local differentiation of the axon to distinct domains, the molecular mechanisms involved are poorly understood. In the present study, we have examined the distribution of axonal components in mice incapable of synthesizing sulfatide by disruption of the galactosylceramide sulfotransferase gene. These mice displayed abnormal paranodal junctions in the CNS and PNS, whereas their compact myelin was preserved. Immunohistochemical analysis demonstrated a decrease in Na+ and K+ channel clusters, altered nodal length, abnormal localization of K+ channel clusters appearing primarily in the presumptive paranodal regions, and diffuse distribution of contactin-associated protein along the internode. Similar abnormalities have been reported previously in mice lacking both galactocerebroside and sulfatide. Interestingly, although no demyelination was observed, these channel clusters decreased markedly with age. The initial timing and the number of Na+ channel clusters formed were normal during development. These results indicate a critical role for sulfatide in proper localization and maintenance of ion channels clusters, whereas they do not appear to be essential for initial cluster formation of Na+ channels.

Reduction-oxidation (redox) regulation has been implicated in the activation of the transcription factor NF-κB. However, the significance and mechanism of the redox regulation remain elusive, mainly due to the technical limitations caused by rapid proton transfer in redox reactions and by the presence of many redox molecules within cells. Here we establish versatile methods for measuring redox states of proteins and their individual cysteine residues in vitro and in vivo, involving thiol-modifying reagents and LC-MS analysis. Using these methods, we demonstrate that the redox state of NF-κB is spatially regulated by its subcellular localization. While the p65 subunit and most cysteine residues of the p50 subunit are reduced similarly in the cytoplasm and in the nucleus, Cys-62 of p50 is highly oxidized in the cytoplasm and strongly reduced in the nucleus. The reduced form of Cys-62 is essential for the DNA binding activity of NF-κB. Several lines of evidence suggest that the redox factor Ref-1 is involved in Cys-62 reduction in the nucleus. We propose that the Ref-1-dependent reduction of p50 in the nucleus is a necessary step for NF-κB activation. This study also provides the first example of a drug that inhibits the redox reaction between two specific proteins. Reduction-oxidation (redox) regulation has been implicated in the activation of the transcription factor NF-κB. However, the significance and mechanism of the redox regulation remain elusive, mainly due to the technical limitations caused by rapid proton transfer in redox reactions and by the presence of many redox molecules within cells. Here we establish versatile methods for measuring redox states of proteins and their individual cysteine residues in vitro and in vivo, involving thiol-modifying reagents and LC-MS analysis. Using these methods, we demonstrate that the redox state of NF-κB is spatially regulated by its subcellular localization. While the p65 subunit and most cysteine residues of the p50 subunit are reduced similarly in the cytoplasm and in the nucleus, Cys-62 of p50 is highly oxidized in the cytoplasm and strongly reduced in the nucleus. The reduced form of Cys-62 is essential for the DNA binding activity of NF-κB. Several lines of evidence suggest that the redox factor Ref-1 is involved in Cys-62 reduction in the nucleus. We propose that the Ref-1-dependent reduction of p50 in the nucleus is a necessary step for NF-κB activation. This study also provides the first example of a drug that inhibits the redox reaction between two specific proteins. The redox states of cysteine residues, which can change reversibly within cells, often greatly influence the various properties of proteins, such as protein stability, chaperone activity, enzymatic activity, and protein structure (1Bader M. Muse W. Ballou D.P. Gassner C. Bardwell J.C. Cell. 1999; 98: 217-227Abstract Full Text Full Text PDF PubMed Scopus (315) Google Scholar, 2Jakob U. Muse W. Eser M. Bardwell J.C. Cell. 1999; 96: 341-352Abstract Full Text Full Text PDF PubMed Scopus (429) Google Scholar, 3Mannick J.B. Hausladen A. Liu L. Hess D.T. Zeng M. Miao Q.X. Kane L.S. Gow A.J. Stamler J.S. Science. 1999; 284: 651-654Crossref PubMed Scopus (706) Google Scholar, 4Tsai B. Rodighiero C. Lencer W.I. Rapoport T.A. Cell. 2001; 104: 937-948Abstract Full Text Full Text PDF PubMed Scopus (414) Google Scholar, 5Eu J.P. Sun J., Xu, L. Stamler J.S. Meissner G. Cell. 2000; 102: 499-509Abstract Full Text Full Text PDF PubMed Scopus (381) Google Scholar). It has also been suggested that several transcription factors bind to their cognate sites in a redox-regulated manner. Well characterized cases include the prokaryotic transcription factors SoxR and OxyR, which function as oxidative stress sensors, their DNA binding activated through oxidation of critical cysteine residues (6Hidalgo E. Ding H. Demple B. Cell. 1997; 88: 121-129Abstract Full Text Full Text PDF PubMed Scopus (128) Google Scholar, 7Zheng M. Aslund F. Storz G. Science. 1998; 279: 1718-1721Crossref PubMed Scopus (979) Google Scholar). In most cases, however, the roles and mechanisms of redox regulation are not fully defined because it is difficult to monitor the alteration of redox states of proteins mainly due to the rapid proton transfer in redox reactions. A few have directly quantified the redox state of cysteine clustered with iron or amounts of oxidized cysteines using physicochemical or biochemical techniques (3Mannick J.B. Hausladen A. Liu L. Hess D.T. Zeng M. Miao Q.X. Kane L.S. Gow A.J. Stamler J.S. Science. 1999; 284: 651-654Crossref PubMed Scopus (706) Google Scholar, 8Demple B. Methods. 1997; 11: 267-278Crossref PubMed Scopus (21) Google Scholar, 9Ding H. Demple B. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 8445-8449Crossref PubMed Scopus (115) Google Scholar), but these methods cannot describe the whole picture of redox states of a protein and are not widely applicable to other proteins. Therefore, most researchers have chosen an indirect way of using cysteine-substitution mutant proteins (3Mannick J.B. Hausladen A. Liu L. Hess D.T. Zeng M. Miao Q.X. Kane L.S. Gow A.J. Stamler J.S. Science. 1999; 284: 651-654Crossref PubMed Scopus (706) Google Scholar, 4Tsai B. Rodighiero C. Lencer W.I. Rapoport T.A. Cell. 2001; 104: 937-948Abstract Full Text Full Text PDF PubMed Scopus (414) Google Scholar, 5Eu J.P. Sun J., Xu, L. Stamler J.S. Meissner G. Cell. 2000; 102: 499-509Abstract Full Text Full Text PDF PubMed Scopus (381) Google Scholar, 7Zheng M. Aslund F. Storz G. Science. 1998; 279: 1718-1721Crossref PubMed Scopus (979) Google Scholar). NF-κB 1The abbreviations used are: NF-κB, nuclear factor κB; IκB, inhibitor of NF-κB; Ref-1, redox factor-1; Trx, thioredoxin; TrxR, thioredoxin-reductase; AP-1, activator protein-1; E3330, (2E)-3-[5-(2,3-dimethoxy-6-methyl-1, 4-benzoquinoyl)]-2-nonyl-2-propenoic acid; PMA, phorbol 12-myristate 13-acetate; F5M, fluorescein-5-maleimide; NEM, N-ethyl-maleimide; DTT, dithiothreitol; TCEP, tris-(2-carboxyethyl)phosphine-hydrochloride; EMSA, electrophoretic mobility shift assay; LC-MS, liquid chromatography mass spectrometry; MI, mass intensity; RNP, ratio of NEM-labeled peptide is a eukaryotic transcription factor that regulates a wide variety of genes involved in immune function and development (10Baeuerle P.A. Baltimore D. Cell. 1996; 87: 13-20Abstract Full Text Full Text PDF PubMed Scopus (2935) Google Scholar). NF-κB is composed of two subunits, p50 and p65, both of which are members of the Rel family of transcription factors. NF-κB normally exists in the cytoplasm, forming an inactive ternary complex with the inhibitor protein IκBα. Following the application of appropriate stimuli, NF-κB is released from IκBα and translocates into the nucleus, where it binds DNA and activates transcription of target genes. Mechanisms of NF-κB activation have been extensively studied; however, it is largely unknown if, and how, the DNA binding step is activated in cells. Some reports have described that the DNA binding activity of NF-κB is regulated by redox potential in vitro(11Toledano M.B. Leonard W.J. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 4328-4332Crossref PubMed Scopus (579) Google Scholar, 12Matthews J.R. Wakasugi N. Virelizier J.L. Yodoi J. Hay R.T. Nucleic Acids Res. 1992; 20: 3821-3830Crossref PubMed Scopus (729) Google Scholar, 13Hayashi T. Ueno Y. Okamoto T. J. Biol. Chem. 1993; 268: 11380-11388Abstract Full Text PDF PubMed Google Scholar). Recently, we reported an antiinflammatory drug that could allow us to solve this issue. The synthetic quinone derivative, (2E)-3-[5-(2,3-dimethoxy-6-methyl-1, 4-benzoquinoyl)]-2-nonyl-2-propenoic acid (E3330), is a novel anti-NF-κB drug that specifically suppresses DNA binding activity of NF-κB but not those of other inflammatory transcription factors, such as activator protein-1 (AP-1) and nuclear factor of activated T cell (NF-AT), in phorbol 12-myristate 13-acetate (PMA)- stimulated Jurkat cells (14Hiramoto M. Shimizu N. Sugimoto K. Tang J. Kawakami Y. Ito M. Aizawa S. Tanaka H. Makino I. Handa H. J. Immunol. 1998; 160: 810-819PubMed Google Scholar). Interestingly, E3330 did not affect the DNA binding activity of purified NF-κB or several steps of NF-κB activation, p65 and nuclear of NF-κB (14Hiramoto M. Shimizu N. Sugimoto K. Tang J. Kawakami Y. Ito M. Aizawa S. Tanaka H. Makino I. Handa H. J. Immunol. 1998; 160: 810-819PubMed Google Scholar). This us to that E3330 target an unknown nuclear factor that the DNA binding activity of NF-κB. with this we purified and redox from Jurkat nuclear as an protein that DNA binding activity of NF-κB in an N. Sugimoto K. Tang J. T. I. M. Aizawa S. M. H. T. F. A. Tanaka H. H. H. Handa H. 2000; PubMed Scopus Google Scholar). the and of the N. Sugimoto K. Tang J. T. I. M. Aizawa S. M. H. T. F. A. Tanaka H. H. H. Handa H. 2000; PubMed Scopus Google Scholar), E3330 is a inhibitor of Ref-1 Ref-1 is a nuclear protein that as a DNA with an activity B. T. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: PubMed Scopus Google Scholar, Nucleic Acids Res. 1991; PubMed Scopus Google Scholar, S. Miao G. F. T. J. 1992; 11: PubMed Scopus Google Scholar). Ref-1 has been reported to of as as other transcription factors and In of AP-1, it by using in vitro cysteine that DNA binding activity of is stimulated by the activity of We have that Ref-1 the DNA binding activity of NF-κB in vitro as as activation in in an N. Sugimoto K. Tang J. T. I. M. Aizawa S. M. H. T. F. A. Tanaka H. H. H. Handa H. 2000; PubMed Scopus Google Scholar). However, few have been to these redox and these in cells. The of many redox such as and it difficult to the specific redox regulation between proteins in eukaryotic cells. In to the with redox vivo, we E3330 as a because E3330 is a inhibitor that specifically inhibits NF-κB activity but not the other transcription factors regulated by Ref-1 as Using thiol-modifying reagents and LC-MS we the redox states of NF-κB its activation step in We evidence that Cys-62 of NF-κB p50 is reduced by Ref-1 in the nucleus and that this reduction is a for NF-κB activation in and from and in to a of and from of p50 and Ref-1 have been described N. Sugimoto K. Tang J. T. I. M. Aizawa S. M. H. T. F. A. Tanaka H. H. H. Handa H. 2000; PubMed Scopus Google Scholar). of by the by reaction into the sites of protein and purified using as by the proteins p50 with for Ref-1 and with for and by an and purified proteins purified from from proteins in of reaction a of in the reactions to the proteins and to the in protein for various the reactions used in electrophoretic mobility shift or The DNA an NF-κB binding as described (14Hiramoto M. Shimizu N. Sugimoto K. Tang J. Kawakami Y. Ito M. Aizawa S. Tanaka H. Makino I. Handa H. J. Immunol. 1998; 160: 810-819PubMed Google Scholar). of proteins, of DNA and of in a and for to as described (14Hiramoto M. Shimizu N. Sugimoto K. Tang J. Kawakami Y. Ito M. Aizawa S. Tanaka H. Makino I. Handa H. J. Immunol. 1998; 160: 810-819PubMed Google Scholar). reduced cysteines in protein with for by the of in and by of the proteins quantified by an of using a with the in vivo, cells in for the steps with F5M, cells by and in cell of and a to a of and for for and to in cell of and a for and reactions by to the and nuclear Following using an to and of proteins as described similarly to that the with and in a of and with in and The liquid chromatography used to protein a with an of using a of acid a of using for and is defined as where and the mass of NEM-labeled and the mass of peptide a a of with to the of within the peptide The from and the of or reduced form of Cys-62 is critical for DNA binding by of p50 and its with of for DNA binding and redox states by and and p50 cysteine to and and into Jurkat cells by with and and of by using p50 and A cell of in and the redox state of proteins, F5M, a with a as a thiol-modifying but not cysteines the where redox reactions can The of is those of other thiol-modifying such as of and Scholar). Using F5M, the of proteins in the reduced state can quantified as an this we a characterized redox reaction between and in vitro A. M. PubMed Scopus Google Scholar). In the presence of specifically the two cysteine residues, and A of oxidized with of for various and reactions for in and the of with the of both and of to residues, did not to a strongly suggest that the reaction is specific and the of the protein redox We have using that Ref-1 DNA binding activity of and but not N. Sugimoto K. Tang J. T. I. M. Aizawa S. M. H. T. F. A. Tanaka H. H. H. Handa H. 2000; PubMed Scopus Google Scholar). suggest that Ref-1 activates DNA binding of NF-κB by the p50 directly Ref-1 we used the and reduced of p50 by p50 with the and the TCEP, as described in the reduced form of p50 but not the oxidized form strongly to the DNA an NF-κB binding oxidized p50 with amounts of Ref-1 or for to or Ref-1 reduced p50 and its DNA binding in the presence of to p50 these The of reduction with the of DNA that redox reactions between Ref-1 and the redox states of both Ref-1 and p50 Ref-1 and oxidized in p50 reduced to of the a of Ref-1 to in We that Ref-1 directly and p50 by a redox It has been but not that of Ref-1 by E3330 through the of its redox activity N. Sugimoto K. Tang J. T. I. M. Aizawa S. M. H. T. F. A. Tanaka H. H. H. Handa H. 2000; PubMed Scopus Google Scholar). with the E3330 Ref-1-dependent activation of DNA binding by oxidized p50 in a E3330 also the Ref-1-dependent reduction of oxidized p50 In E3330 did not affect redox states or DNA binding activity of the reduced form of these we that E3330 is by the of Ref-1 redox The is a way to in the redox of proteins. However, it not in cases where proteins reduced cysteines of which a is to redox regulation because of cysteines a of p50 has cysteine residues and cysteine residues of p50 that are reduced by Ref-1, we the The of reduced cysteines with derivative, NEM, of NEM-labeled proteins by and LC-MS for the of NEM-labeled and because could not fully from liquid due to the as a of not the mass of NEM-labeled and and the of reduced cysteine residues using the or the ratio of NEM-labeled peptide the in we that residues reduced by and Ref-1 p50 oxidized with with or Ref-1 and as described of p50 include a cysteine and two with two cysteine residues A mass of the oxidized p50 a of Cys-62 to fully oxidized because the peptide with or Ref-1 in the NEM-labeled peptide that Cys-62 reduced by these The from their mass suggest that of p50 with or Ref-1 in or reduction of Cys-62 of p50 from of mass in a by LC-MS analysis. for these are in B. two for and the cysteine residues reduced by Ref-1 by however, the reduction of Cys-62 by Ref-1 and highly to E3330 for an of reactions The DNA binding activity of p50 with the redox states of that Cys-62 a in DNA did not the cysteine residues of of the cysteine residues the with this that of and which not reduced by in are in the p50 protein G. 1998; PubMed Scopus Google Scholar). The not the in to other cysteines in redox regulation of this we and for of cysteine residues and to and chosen because it a reduced form of of the described and by J.R. Wakasugi N. Virelizier J.L. Yodoi J. Hay R.T. Nucleic Acids Res. 1992; 20: 3821-3830Crossref PubMed Scopus (729) Google Scholar, M.B. D. F. Leonard W.J. Cell. Biol. 1993; PubMed Google Scholar). for DNA binding of and not oxidized and strongly stimulated by in a In DNA binding in its oxidized form and not by by the redox states of the other cysteine The of are with those of reports J.R. Wakasugi N. Virelizier J.L. Yodoi J. Hay R.T. Nucleic Acids Res. 1992; 20: 3821-3830Crossref PubMed Scopus (729) Google Scholar, M.B. D. F. Leonard W.J. Cell. Biol. 1993; PubMed Google J.R. W. G. Hay R.T. Nucleic Acids Res. 1993; PubMed Scopus Google Scholar). it is that Cys-62 is for reduction and activation of Using these methods, we the that the redox state of NF-κB is regulated the activation We a Jurkat cell p50 to and the to a of NF-κB complex from cells. similarly to it a ternary complex with p65 and IκBα in the cytoplasm, into the nucleus with and DNA as a with p65 in a not the redox state of NF-κB in cells, we the as cells with or for in F5M, and into and nuclear these NF-κB with and amounts and of and p65 by and of the by the to its protein and of and p65 in cells as a of in the nucleus of cells, the analysis. the protein strongly in the nucleus in the cytoplasm of cells and In the of p65 in both the cytoplasm and nucleus. of cells nuclear p50 and p65 protein but did not affect their redox of subcellular that p50 is reduced into the nucleus and that this reduction of this is the first redox states of a protein in the nucleus and is the for the reduction of p50 in the the various redox proteins, and Ref-1 have been implicated in the of NF-κB activity J.R. Wakasugi N. Virelizier J.L. Yodoi J. Hay R.T. Nucleic Acids Res. 1992; 20: 3821-3830Crossref PubMed Scopus (729) Google Scholar, 13Hayashi T. Ueno Y. Okamoto T. J. Biol. Chem. 1993; 268: 11380-11388Abstract Full Text PDF PubMed Google K. K. K. Sun S. K. PubMed Scopus Google Scholar). We E3330 to the of Ref-1 in the reduction of p50 in the nucleus. in of cells with of E3330 to p50 reduction in the nucleus, but not in the In E3330 the redox states of p65 that Ref-1 is involved in p50 reduction in the nucleus but not in the reduction of or nuclear We the redox states of individual cysteine residues of p50 using a and LC-MS analysis. Following with E3330 and PMA, cells with and into and nuclear to chromatography and chromatography to amounts of from the nuclear and this with and to LC-MS, and the similarly to A the mass of the In the cytoplasm of or cells, a of p50 Cys-62 as from the mass of peptide which that of the NEM-labeled peptide In p50 in the cell nucleus reduced from the mass of NEM-labeled and the similarly by LC-MS and for these are in B. that or of the and residues are reduced of the subcellular of NF-κB or the presence of or In the of Cys-62 reduction to in the cytoplasm and in the nucleus. reduction is in the cytoplasm and in the nucleus. that reduced state of p50 in A is due to the reduction of these cysteine the reduction of Cys-62 by of the cells with E3330, in a reduction that in the cytoplasm A and In the reduction of in the nucleus not by It is that Ref-1 a in the reduction of Cys-62 in the nucleus but not of the other cysteine residues of with the that E3330 inhibits activation (14Hiramoto M. Shimizu N. Sugimoto K. Tang J. Kawakami Y. Ito M. Aizawa S. Tanaka H. Makino I. Handa H. J. Immunol. 1998; 160: 810-819PubMed Google Scholar), strongly suggest that reduction of p50 Cys-62 in the nucleus is a for NF-κB activation. We that Ref-1 specifically reduced Cys-62 of which to activation of NF-κB DNA binding in vitro and in We also that and the redox of have implicated in the redox regulation of NF-κB J.R. Wakasugi N. Virelizier J.L. Yodoi J. Hay R.T. Nucleic Acids Res. 1992; 20: 3821-3830Crossref PubMed Scopus (729) Google Scholar, 13Hayashi T. Ueno Y. Okamoto T. J. Biol. Chem. 1993; 268: 11380-11388Abstract Full Text PDF PubMed Google Scholar, K. K. K. Sun S. K. PubMed Scopus Google Scholar). However, several lines of evidence suggest that Ref-1 a in p50 reduction in In a of Ref-1, but not of Trx, reduced and activated p50 in that Ref-1 a potential used a of and a or to activation of NF-κB J.R. Wakasugi N. Virelizier J.L. Yodoi J. Hay R.T. Nucleic Acids Res. 1992; 20: 3821-3830Crossref PubMed Scopus (729) Google Scholar, K. K. K. Sun S. K. PubMed Scopus Google Scholar). In in the cytoplasm K. M. S. A. K. Yodoi J. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: PubMed Scopus Google Scholar). that Ref-1 exists cell or to to exists molecules cell or to in the nucleus of Jurkat or cells. and H. It reported that translocates into the nucleus of cells with or K. M. S. A. K. Yodoi J. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: PubMed Scopus Google Scholar, K. M. Y. H. J. K. Yodoi J. J. Biol. Chem. 1999; Full Text Full Text PDF PubMed Scopus Google however, we did not change in not p50 in the nucleus reduced within and because this reduction strongly by E3330 A and it that directly p50 in the nucleus, to NF-κB activation by Ref-1 redox activity K. M. S. A. K. Yodoi J. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: PubMed Scopus Google Scholar, A. K. S. A. Yodoi J. D. Res. 2000; Google or by in the cytoplasm J. K. T. K. Yodoi J. T. K. 2000; PubMed Scopus Google Scholar). are with a that of Ref-1 with the activation of DNA binding by NF-κB, of Ref-1 by an DNA binding activity of NF-κB in D. A. D. L. E. A. A. J. Biol. Chem. 2001; Full Text Full Text PDF PubMed Scopus Google Scholar). Ref-1 p50 but not its p50 and p65 the Rel their and The which two DNA binding and is for of the Ref-1 We have using that Ref-1 with p50 but not with p65 N. Sugimoto K. Tang J. T. I. M. Aizawa S. M. H. T. F. A. Tanaka H. H. H. Handa H. 2000; PubMed Scopus Google Scholar). the p50 the target of Ref-1, is highly other members of the Rel it is that the is necessary to the specific between Ref-1 and We also that the redox states of p50 are regulated by its subcellular the cysteine residues, Cys-62 and are of reduction their to E3330, it is that Cys-62 and are reduced by Ref-1 and redox in the nucleus. are these residues oxidized strongly in the Ref-1, which is for Cys-62 reduction in the nucleus, is both in the cytoplasm and in the nucleus not Ref-1 redox activity with its and Ref-1 of p50 This is with of in that Ref-1 is in a oxidized state in the cytoplasm redox molecules for reduction as in the nucleus. reduction of these residues in the cytoplasm by the presence of In this it that within the T. S. G. Cell. 1998; Full Text Full Text PDF PubMed Scopus Google Scholar, Cell. 1998; Full Text Full Text PDF PubMed Scopus Google Scholar). binding to NF-κB directly the of redox molecules to the target cysteines or its change T. S. G. Cell. 1998; Full Text Full Text PDF PubMed Scopus Google such that the redox molecules cannot the target The two are not and both to the of Cys-62 and reduction in the Using LC-MS, we that activation of DNA binding by NF-κB is caused by the reduction of p50 is the for the redox regulation of to the structure of NF-κB in a complex with Cys-62 is in the DNA G. 1998; PubMed Scopus Google Scholar, G. G. S. PubMed Scopus Google Scholar, M. PubMed Scopus Google Scholar). the a that of the is of the reduced form of and The of these residues is by the that are the Rel oxidation of Cys-62 have a the structure and DNA of this Cys-62 by to highly to the DNA Cys-62 is by This the of Cys-62 highly and to with this Cys-62 strongly oxidized in the cytoplasm and in the nucleus in the cells, Cys-62 oxidized in the Ref-1, the nucleus to a oxidative for Cys-62 the the other cysteine residues did not of redox states the of Cys-62 oxidation from in within its subcellular We that binding to NF-κB not Ref-1-dependent reduction but also Cys-62 from by the of residues In this the DNA such that Cys-62 is highly to Ref-1 to this cysteine reduced in the nucleus. of not a of p50 these residues, this a T. S. G. Cell. 1998; Full Text Full Text PDF PubMed Scopus Google Scholar, Cell. 1998; Full Text Full Text PDF PubMed Scopus Google Scholar). of its the of Cys-62 is to the redox regulation of NF-κB. of oxidation Cys-62 in We could not it because the oxidized of cysteine are reduced the step in the This reduction which is to to of other and to the of the critical for of p50 not However, the of by using other reagents and reactions by and the of by using other and of cysteine include or acid and is a involving and to oxidation of p50 Cys-62 J.R. M. Hay R.T. Nucleic Acids Res. 1996; PubMed Scopus Google Scholar, A. Res. 1997; PubMed Scopus Google Scholar, S. J. 1997; PubMed Scopus Google Scholar, A. C. J. Immunol. 1999; Google Scholar). In this we have methods for measuring the redox state of a protein using We that the by these methods the redox states of p50 because of the of this study is that Cys-62 is the redox target within cells. This with a of reports its by indirect using in vitro J.R. Wakasugi N. Virelizier J.L. Yodoi J. Hay R.T. Nucleic Acids Res. 1992; 20: 3821-3830Crossref PubMed Scopus (729) Google Scholar, of and Scholar, A. M. PubMed Scopus Google Scholar). that the redox states of p50 could change the of the protein from the cells. However, this is not because we did not change in the redox states of p50 that oxidized or reduced using the techniques as used for Jurkat cell not It that is in the by these affect the of the LC-MS by the physicochemical of by the and the the can the reduction of cysteines the of or is not greatly by of the protein within the cell or by of the cysteine within the it cannot that the of reduced the by several the we that this is not a for because which is in the p50 protein G. 1998; PubMed Scopus Google Scholar), by an of in and are various or the of proteins, the reduced cysteines In the methods allow us to and to a that can by other In the methods described are applicable to Some include the of the as and the of a of proteins from cells for LC-MS analysis. In this a of cells used for analysis. The protein amounts for these can greatly by the of LC-MS analysis. these methods are the versatile of the redox states of cysteines in vitro and in and an for the study of redox We for We Tang and for

Dorsoventral patterning of the vertebrate nervous system is achieved by the combined activity of morphogenetic signals secreted from dorsal and ventral signalling centres. The Shh/Gli pathway plays a major role in patterning the ventral neural tube; however, the molecular mechanisms that limit target gene responses to specific progenitor domains remain unclear. Here, we show that Wnt1/Wnt3a, by signalling through the canonical beta-catenin/Tcf pathway, control expression of dorsal genes and suppression of the ventral programme, and that this role in DV patterning depends on Gli activity. Additionally, we show that Gli3 expression is controlled by Wnt activity. Identification and characterization of highly conserved non-coding DNA regions around the human Gli3 gene revealed the presence of transcriptionally active Tcf-binding sequences. These indicated that dorsal Gli3 expression might be directly regulated by canonical Wnt activity. In turn, Gli3, by acting as a transcriptional repressor, restricted graded Shh/Gli ventral activity to properly pattern the spinal cord.

Large single crystals of the chiral ferrimagnet [{Cr(CN)6}{Mn(S)-pnH(H2O)}](H2O) can be recovered from the reaction between K3[Cr(CN)6], Mn(ClO4)2, and (S)-1,2-diaminopropane dihydrochloride (see picture). Magnetic measurements of the complex show that MnII and CrIII ions interact ferrimagnetically, with a magnetic transition occurring at 38 K. Supporting information for this article is available on the WWW under http://www.wiley-vch.de/contents/jc_2002/2003/z51888_s.pdf or from the author. Please note: The publisher is not responsible for the content or functionality of any supporting information supplied by the authors. Any queries (other than missing content) should be directed to the corresponding author for the article.

In the endoplasmic reticulum (ER), lectins and processing enzymes are involved in quality control of newly synthesized proteins for productive folding as well as in the ER-associated degradation (ERAD) of misfolded proteins. ER quality control requires the recognition and modification of the N-linked oligosaccharides attached to glycoproteins. Mannose trimming from the N-glycans plays an important role in targeting of misfolded glycoproteins for ERAD. Recently, two mammalian lectins, OS-9 and XTP3-B, which contain mannose 6-phosphate receptor homology domains, were reported to be involved in ER quality control. Here, we examined the requirement for human OS-9 (hOS-9) lectin activity in degradation of the glycosylated ERAD substrate NHK, a genetic variant of alpha1-antitrypsin. Using frontal affinity chromatography, we demonstrated that the recombinant hOS-9 mannose 6-phosphate receptor homology domain specifically binds N-glycans lacking the terminal mannose from the C branch in vitro. To examine the specificity of OS-9 recognition of N-glycans in vivo, we modified the oligosaccharide structures on NHK by overexpressing ER alpha1,2-mannosidase I or EDEM3 and examined the effect of these modifications on NHK degradation in combination with small interfering RNA-mediated knockdown of hOS-9. The ability of hOS-9 to enhance glycoprotein ERAD depended on the N-glycan structures on NHK, consistent with the frontal affinity chromatography results. Thus, we propose a model for mannose trimming and the requirement for hOS-9 lectin activity in glycoprotein ERAD in which N-glycans lacking the terminal mannose from the C branch are recognized by hOS-9 and targeted for degradation.

A 2 in. diam single crystal of lithium tetraborate (Li2B4O7) was successfully grown by the Czochralski method. The crystal was free from macrodefects and had a dislocation density as low as 100/cm2. It had an excellent homogeneity of the refractive index and a wide transparency down to 170 nm. The optical damage threshold was 40 GW/cm2. Second-harmonic generation and sum frequency generation were investigated in association with the generation of the fourth and fifth harmonics of a Q-switched Nd:YAG laser. The conversion efficiency of the second-harmonic generation from the green (532 nm) light was 20%.

Abstract A computer‐operated spectrograph was recently built at Okazaki, Japan. Different specimens can be placed on a horseshoe‐shaped focal curve (10 m long) covering a wavelength range of 250 to 1000 nm so they can be irradiated simultaneously. The linear dispersion is about 0.8 nm/cm. The photon fluence rate on the focal curve is 5 x 10 15 . photons x cm ‐2 x s ‐1 at 300nm and 1 x 10 16 photons x cm ‐2 x s ‐1 at 600 and at 900 nm. The spectral half width is 5.5 nm or less on the focal curve. The stray light content is about 10 ‐5 of the main peak at the peak wavelength ± 100 nm. Specimens are set in microcomputer‐controlled threshold boxes so that wavelengths, photon fluence rates, photon fluences and timing of irradiations are controlled automatically according to a pre‐programmed schedule. An optical fiber system is also provided for remote irradiations.

F1-ATPase is a rotary motor made of a single protein molecule. Its rotation is driven by free energy obtained by ATP hydrolysis. In vivo, another motor, Fo, presumably rotates the F1 motor in the reverse direction, reversing also the chemical reaction in F1 to let it synthesize ATP. Here we attempt to answer two related questions, How is free energy obtained by ATP hydrolysis converted to the mechanical work of rotation, and how is mechanical work done on F1 converted to free energy to produce ATP? After summarizing single-molecule observations of F1 rotation, we introduce a toy model and discuss its free-energy diagrams to possibly answer the above questions. We also discuss the efficiency of molecular motors in general.