Biomedical subjects

Daisuke Yamazaki

Publications and source records attributed to Daisuke Yamazaki.

14 recordsLinked to original sources

PubMed2005

Rac-WAVE2 signaling is involved in the invasive and metastatic phenotypes of murine melanoma cells.

WAVEs (WASP-family verprolin-homologous proteins) regulate the actin cytoskeleton through activation of Arp2/3 complex. As cell motility is regulated by actin cytoskeleton rearrangement and is required for tumor invasion and metastasis, blocking actin polymerization may be an effective strategy to prevent tumor dissemination. We show that WAVEs, especially WAVE2, are essential for invasion and metastasis of melanoma cells. Malignant B16F10 mouse melanoma cells expressed more WAVE1 and WAVE2 proteins and showed higher Rac activity than B16 parental cells, which are neither invasive nor metastatic. The effect of WAVE2 silencing by RNA interference (RNAi) on the highly invasive nature of B16F10 cells was more dramatic than that of WAVE1 RNAi. Membrane ruffling, cell motility, invasion into the extracellular matrix, and pulmonary metastasis of B16F10 cells were suppressed by WAVE2 RNAi. WAVE2 RNAi also had a profound effect on invasion induced by a constitutively active form of Rac (RacCA). In addition, ectopic expression of both RacCA and WAVE2 in B16 cells resulted in further increase in the invasiveness than that observed in B16 cells expressing only RacCA. Thus, WAVE2 acts as the primary effector downstream of Rac to achieve invasion and metastasis, suggesting that suppression of WAVE2 activity holds a promise for preventing cancer invasion and metastasis.

Actins↗

PubMed2004

Pyrimidine-core extended pi-systems: general synthesis and interesting fluorescent properties.

We have developed a simple but powerful synthetic strategy that permits the assembly of pi-systems onto a pyrimidine core in a programmable and diversity-oriented format. The nucleophilic addition of ArLi to 2-methylthiopyrimidine, followed by 2,3-dichloro-5,6-dicyano-1,4-benzoquinone (DDQ) oxidation, resulted in the production of 4-aryl-2-methylthiopyrimidines. The iterative reaction sequence then gave 4,6-diaryl-2-methylthiopyrimidines. The resulting adduct was further allowed to react with ArMgBr under the catalytic influence of NiCl2(dppe) to afford 2,4,6-triarylpyrimidines. By following this synthetic scheme, interesting pyrimidine-core pi-systems were rapidly constructed in a programmable fashion. The successful discovery of a number of interesting fluorescent materials and properties (e.g., solvatofluorochromism) speaks well for the potential of our platform strategy in the development of functional organic materials.

Journal Article↗

PubMed2004

Radical cation of dibenzothiophene fully annelated with bicyclo[2.2.2]octene units: X-ray crystal structure and electronic properties.

New dibenzothiophene 2 fully annelated with bicyclo[2.2.2]octene units was synthesized and oxidized to stable radical cation salt 2(*+)SbCl(6)(-), whose structure was determined by X-ray crystallography. Although the intrinsic electronic structure of 2(*+) is predicted to be close to structure A, an interaction with the counteranion makes structure B contribute significantly. A part of the salt 2(*+)SbCl(6)(-) underwent rearrangement to arenium ion 6(+), whose structure was also clarified by X-ray crystallography.

Journal Article↗

PubMed2004

N-WASP and WAVE2 acting downstream of phosphatidylinositol 3-kinase are required for myogenic cell migration induced by hepatocyte growth factor.

During skeletal muscle regeneration caused by injury, muscle satellite cells proliferate and migrate toward the site of muscle injury. This migration is mainly induced by hepatocyte growth factor (HGF) secreted by intact myofibers and also released from injured muscle. However, the intracellular machinery for the satellite cell migration has not been elucidated. To examine the mechanisms of satellite cell migration, we utilized satellite cell-derived mouse C2C12 skeletal muscle cells. HGF induced reorganization of actin cytoskeleton to form lamellipodia in C2C12 myoblasts. HGF treatment facilitated both nondirectional migration of the myoblasts in phagokinetic track assay and directional chemotactic migration toward HGF in a three-dimensional migration chamber assay. Endogenous N-WASP and WAVE2 were concentrated in the lamellipodia at the leading edge of the migrating cells. Moreover, exogenous expression of wild-type N-WASP or WAVE2 promoted lamellipodial formation and migration. By contrast, expression of the dominant-negative mutant of N-WASP or WAVE2 and knockdown of N-WASP or WAVE2 expression by the RNA interference prevented the HGF-induced lamellipodial formation and migration. When the cells were treated with LY294002, an inhibitor of phosphatidylinositol 3-kinase, the HGF-induced lamellipodial formation and migration were abrogated. These results imply that both N-WASP and WAVE2, which are activated downstream of phosphati-dylinositol 3-kinase, are required for the migration through the lamellipodial formation of C2C12 cells induced by HGF.

Actins↗

PubMed2004

WAVE/Scars in platelets.

Using specific antibodies against isoforms of WAVE (WASP [Wiskott-Aldrich syndrome protein] family Verprolin-homologous protein, also called Scar), we demonstrated that human platelets express all 3 isoforms. With the use of an in vitro pull-down technique, the src homology 3 (SH3) domain of insulin receptor substrate p53 (IRSp53) precipitated WAVE2 from platelet lysates more efficiently than did profilin I. The opposite was true for WAVE1, and neither precipitated WAVE3, suggesting that WAVE isoforms have different affinities to these ligands, while the SH3 domain of abl binds to all 3 isoforms. The 3 WAVE isoforms were distributed in the actin-rich Triton X-100-insoluble pellets following platelet aggregation induced by thrombin receptor-activating peptide. We also found that all 3 WAVE isoforms are substrates for calpain in vivo and in vitro. Although portions of these 3 isoforms were commonly distributed in the actin- and actin-related protein 2 and 3 (Arp2/3)-rich edge of the lamellipodia in spreading platelets, only WAVE2 remained in the cell fringe following detergent extraction or fixation of the cells. Finally, by mass spectrometry, we found that the proteins, which reportedly interact with WAVE/Scars, are present in platelets. These data suggest that the 3 WAVE isoforms exhibit common and distinct features and may potentially be involved in the regulation of actin cytoskeleton in platelets.

Actin Cytoskeleton↗

PubMed2004

PtdIns(3,4,5)P3 binding is necessary for WAVE2-induced formation of lamellipodia.

Polarized cell movement is triggered by the development of a PtdIns(3,4,5)P(3) gradient at the membrane, which is followed by rearrangement of the actin cytoskeleton. The WASP family verprolin homologous protein (WAVE) is essential for lamellipodium formation at the leading edge by activating the Arp2/3 complex downstream of Rac GTPase. Here, we report that WAVE2 binds to PtdIns(3,4,5)P(3) through its basic domain. The amino-terminal portion of WAVE2, which includes the PtdIns(3,4,5)P(3)-binding sequence, was localized at the leading edge of lamellipodia induced by an active form of Rac (RacDA) or by treatment with platelet-derived growth factor (PDGF). Production of PtdIns(3,4,5)P(3) at the cell membrane by myristoylated phosphatidylinositol-3-OH kinase (PI(3)K) is sufficient to recruit WAVE2 in the presence of dominant-negative Rac and latrunculin, demonstrating that PtdIns(3,4,5)P(3) alone is able to recruit WAVE2. Expression of a full-length mutant of WAVE2 that lacks the lipid-binding activity inhibited proper formation of lamellipodia induced by RacDA. These results suggest that one of the products of PI(3)K, PtdIns(3,4,5)P(3), recruits WAVE2 to the polarized membrane and that this recruitment is essential for lamellipodium formation at the leading edge.

Amino Acid Sequence↗

PubMed2004

Crystal structures and spectroscopic characterization of radical cations and dications of oligothiophenes stabilized by annelation with bicyclo[2.2.2]octene units: sterically segregated cationic oligothiophenes.

The remarkably stable SbF(6)(-) salts of the radical cations of bithiophene 1(2T) and terthiophene 1(3T), completely surrounded by bicyclo[2.2.2]octene (BCO) units, were obtained by one-electron oxidation of the neutral precursors with NO(+)SbF(6)(-), and their solid-state structures were determined by X-ray crystallography. In these radical cations, the presence of quinoidal character was apparent, as shown by the increased planarity and by comparison of the bond lengths with those of the neutral precursors. The shortest intermolecular pi-pi distances in the crystal structure of 1(2T)(*)(+)SbF(6)(-) (distance between two sp(2) carbon atoms, 4.89 A) and 1(3T)(*)(+)SbF(6)(-) (distance between an sp(2) carbon and a sulfur atom, 3.58 A) were found to be longer than the sums of the van der Waals radii of the corresponding atoms. In accord with this, no apparent change was observed in ESR and UV-vis-NIR spectra of solutions of 1(2T)(*)(+) and 1(3T)(*)(+) upon lowering the temperature, indicating that the pi- (or sigma-) dimer formation is inhibited in solution as well as in the solid state. The dications 1(2T)(2+) and 1(3T)(2+) were generated with the stronger oxidant SbF(5) in CH(2)Cl(2) at -40 degrees C and characterized by NMR spectroscopy. In the (1)H NMR spectra, two conformers were observed for each dication of both 1(2T)(2+) (transoid (t) and cisoid (c)) and 1(3T)(2+) (t,t and c,t) at room temperature due to the high rotational barrier around the inter-ring bond(s) between thiophene rings, which was caused by the enhanced double bond character of these bonds following two-electron oxidation. This is supported by DFT calculations (B3LYP/6-31G(d)), which predicted the rotational barriers in the dications of unsubstituted bithiophene and terthiophene to be 27.6 and 22.9 kcal mol(-)(1), respectively. In the case of quaterthiophene and sexithiophene surrounded by BCO frameworks 1(4T) and 1(6T), oxidation with even one molar equivalent of NO(+)SbF(6)(-) afforded the dication salts 1(4T)(2+)2SbF(6)(-) and 1(6T)(2+)2SbF(6)(-), which were isolated as stable single crystals and allowed the X-ray crystallography. In their crystal structures, the cationic pi-systems became planar again due to the great contribution of quinoidal resonance structures, and the pi-systems, which were arrayed in a parallel geometry, were also segregated by the steric effect of BCO units. The degree and tendency of changes in the bond lengths of thiophene rings of 1(4T)(2+) and 1(6T)(2+) as compared with neutral precursors were similar to those of 1(2T)(*)(+)SbF(6)(-) and 1(3T)(*)(+)SbF(6)(-), respectively, implying that the contribution of quinoidal character is modulated by the amount of positive charge per thiophene unit.

Journal Article↗

PubMed2004

Target proteins of the cytosolic thioredoxins in Arabidopsis thaliana.

Possible target proteins of cytosolic thioredoxin in higher plants have been investigated in the cell lysate of dark-grown Arabidopsis thaliana whole tissues. We immobilized a mutant of cytosolic thioredoxin, in which an internal cysteine at the active site was substituted with serine, on CNBr activated resin, and used the resin for the thioredoxin-affinity chromatography. By using this resin, the target proteins for thioredoxin in the higher plant cytosol were efficiently acquired. The obtained proteins were separated by two-dimensional gel electrophoresis and analyzed by matrix-assisted laser desorption ionization time-of-flight mass spectrometry. Thus we have identified proteins of the anti-oxidative stress system proteins (ascorbate peroxidase, germin-like protein, and monomeric type II peroxiredoxin), proteins involved in protein biosynthesis (elongation factor-2 and eukaryotic translation initiation factor 4A), proteins involved in protein degradation (the regulatory subunit of 26S proteasome), and several metabolic enzymes (alcohol dehydrogenase, fructose 1,6-bis phosphate aldolase-like protein, cytosolic glyceraldehyde 3-phosphate dehydrogenase, cytosolic malate dehydrogenase, and vitamin B(12)-independent methionine synthase) together with some chloroplast proteins (chaperonin 60-alpha and 60-beta, heat shock protein 70, and glutamine synthase). The results in this study and recent proteomics studies on the target proteins of chloroplast thioredoxin indicate the versatility and the physiological significance of thioredoxin as reductant in plant cell.

Antioxidants↗

PubMed2003

Synthesis and properties of novel oligothiophenes surrounded by bicyclo[2.2.2]octene frameworks.

Novel oligothiophenes surrounded by bicyclo[2.2.2]octene (abbreviated as BCO) frameworks ranging from dimer to hexamer, 1(nT) (n = 2, 3, 4, 6), were prepared, and their structures and electronic properties were investigated. Dimer 1(2T) was synthesized by oxidative coupling of the 2-lithiated monomer generated from 4,5-BCO-annelated 2-bromothiophene 8 with CuCl2 in 76% yield. Trimer 1(3T) and tetramer 1(4T) were synthesized by Stille coupling of 2,5-dibromo-3,4-BCO-annelated thiophene 4 and of the 5,5'-dibromo derivative of bis(3,4-BCO-thiophen-2-yl) 10 with 2-stannylated 4,5-BCO-annelated thiophene 9 in 41% and 46% yield, respectively. Hexamer 1(6T) was synthesized by oxidative coupling of terthiophene 12, tris-annelated with BCO units, in 81% yield. X-ray crystallographic studies showed that the thiophene rings in 1(2T) and 1(3T) are rotated around the inter-ring C-C bond(s) with the C=C-C=C dihedral angles of -174.3(5) degrees for 1(2T) and -149.7(3) degrees and 34.4(3) degrees for 1(3T). In the crystal structures of 1(2T) and 1(3T), no pi-stacking was observed as expected from the steric effect of the BCO units. Theoretical calculations for 1(2T) and 1(3T) at the B3LYP/6-31G(d) level indicated that the annelation with BCO units either at the 2,3- or 3,4-positions of thiophene rings raises both the KS HOMO and LUMO levels. In the electronic absorption spectra of 1, the longest wavelength absorption band corresponding to the pi-pi transition is bathochromically shifted with the increase in absorption intensity as the number of thiophene rings increases, and the absorption of the polythiophene 1 with infinite length was predicted to be 419 nm. The cyclic voltammetry of 1 in CH2Cl2 at -78 degrees C (2T) or at room temperature (3T, 4T, 6T) showed two reversible oxidation waves, indicating that the radical cation and dication of 1 are stable under these conditions.

Journal Article↗

PubMed2003

WAVE2 is required for directed cell migration and cardiovascular development.

WAVE2, a protein related to Wiskott-Aldrich syndrome protein, is crucial for Rac-induced membrane ruffling, which is important in cell motility. Cell movement is essential for morphogenesis, but it is unclear how cell movement is regulated or related to morphogenesis. Here we show the physiological functions of WAVE2 by disruption of the WAVE2 gene in mice. WAVE2 was expressed predominantly in vascular endothelial cells during embryogenesis. WAVE2-/- embryos showed haemorrhages and died at about embryonic day 10. Deficiency in WAVE2 had no significant effect on vasculogenesis, but it decreased sprouting and branching of endothelial cells from existing vessels during angiogenesis. In WAVE2-/- endothelial cells, cell polarity formed in response to vascular endothelial growth factor, but the formation of lamellipodia at leading edges and capillaries was severely impaired. These findings indicate that WAVE2-regulated actin reorganization might be required for proper cell movement and that a lack of functional WAVE2 impairs angiogenesis in vivo.

Animals↗

PubMed2003

Synthesis, structure, and dynamic behavior of cyclopentadienyl-lithium, -sodium, and -potassium annelated with bicyclo[2.2.2]octene units: a systematic study on site exchange of alkali metals on a cyclopentadienyl ring in tetrahydrofuran.

Novel cyclopentadienyl (Cp)-alkali metal complexes 1-M and 2-M (M = Li, Na, K), in which the Cp ring is annelated with two bicyclo[2.2.2]octene units and substituted with a phenyl group for 1 and a tert-butyl group for 2, were synthesized, and their structures and dynamic behaviors were investigated by means of X-ray crystallography, dynamic (13)C NMR, and DFT calculations. The X-ray crystallography results indicated that 1-Li, 1-Na, and 2-Na form monomeric contact ion pairs (CIP) with three THF molecules coordinated to the metal atom. Also, in THF-d(8), all of the 1-M and 2-M form monomeric CIP in the ground state. However, variable-temperature (13)C NMR measurements of 1-M and 2-M in THF-d(8) demonstrated dynamic behavior in which the metal ion exchanges positions between the upper and lower faces of the Cp ring. From a study of the concentration dependence of the dynamic behavior, the exchange was found to proceed principally as an intramolecular process at concentration ranges lower than 0.2 M. The experimentally observed deltaG values for the intramolecular exchange process for all the 1-M and 2-M (except for 2-Li, whose intramolecular process was too slow to observe) were found to be quite similar in THF-d(8) solution and to fall within the range of 12-14 kcal mol(-)(1). Within this range, a tendency was observed for the deltaG values to increase as the size of the metal decreased. Theoretical calculations (B3LYP/6-31G(d)) afforded considerably large values as the gas-phase dissociation energy for 1-M (162.7 kcal mol(-)(1) for M = Li; 131.6 kcal mol(-)(1) for M = Na; 110.9 kcal mol(-)(1) for M = K) and for 2-M (170.0 kcal mol(-)(1) for M = Li; 137.5 kcal mol(-)(1) for M = Na; 115.4 kcal mol(-)(1) for M = K). These values should be compensated for by a decrease in the solvation energies for the metal ions with increasing size, as exemplified by the calculated solvation energy for M(+)(Me(2)O)(4), which serves as a model for metal ions solvated with four molecules of THF (-122.9 kcal mol(-)(1) for M = Li; -94.7 kcal mol(-)(1) for M = Na; -67.7 kcal mol(-)(1) for M = K). This compensation results in a small difference in the overall energy for dissociation of 1-M or 2-M in ethereal solutions, thus supporting the similar deltaG values observed for the intramolecular metal exchange.

Journal Article↗

PubMed2003

Palladium-catalyzed rearrangement/arylation of 2-allyloxypyridine leading to the synthesis of N-substituted 2-pyridones.

[reaction: see text] The Pd-catalyzed one-pot rearrangement/arylation of 2-allyloxypyridine is described. The catalyst/base combination of Pd[P(t-Bu)(3)](2)/Ag(2)CO(3) was found to be optimal for this one-pot rearrangement/arylation. The initial rearrangement of 2-allyloxypyridine was found to be catalyzed by both Pd(0) and Pd(II) complexes with different mechanisms.

Journal Article↗

PubMed2003

Differential roles of WAVE1 and WAVE2 in dorsal and peripheral ruffle formation for fibroblast cell migration.

Cell migration is driven by actin polymerization at the leading edge of lamellipodia, where WASP family verprolin-homologous proteins (WAVEs) activate Arp2/3 complex. When fibroblasts are stimulated with PDGF, formation of peripheral ruffles precedes that of dorsal ruffles in lamellipodia. Here, we show that WAVE2 deficiency impairs peripheral ruffle formation and WAVE1 deficiency impairs dorsal ruffle formation. During directed cell migration in the absence of extracellular matrix (ECM), cells migrate with peripheral ruffles at the leading edge and WAVE2, but not WAVE1, is essential. In contrast, both WAVE1 and WAVE2 are essential for invading migration into ECM, suggesting that the leading edge in ECM has characteristics of both ruffles. WAVE1 is colocalized with ECM-degrading enzyme MMP-2 in dorsal ruffles, and WAVE1-, but not WAVE2-, dependent migration requires MMP activity. Thus, WAVE2 is essential for leading edge extension for directed migration in general and WAVE1 is essential in MMP-dependent migration in ECM.

Actin Cytoskeleton↗

PubMed1998

Intracellular chitinase gene from Rhizopus oligosporus: molecular cloning and characterization.

Multiple chitinases have been found in hyphae of filamentous fungi, which are presumed to have various functions during hyphal growth. Here it is reported, for the first time, the primary structure of one such intracellular chitinase, named chitinase III, from Rhizopus oligosporus, a zygomycete filamentous fungus. Chitinase III was purified to homogeneity from actively growing mycelia of R. oligosporus using three steps of column chromatography. Its molecular mass was 43.5 kDa and the pH optimum was 6.0 when p-nitrophenyl N,N',N"-beta-D-triacetylchitotrioside was used as a substrate. Chitinase III also hydrolysed chromogenic derivatives of chitobiose, but had no N-acetylglucosaminidase activity. The gene encoding chitinase III (chi3) was cloned using PCR with degenerate oligonucleotide primers from the partial amino acid sequence of the enzyme. The deduced amino acid sequence of chi3 was similar to that of bacterial chitinases and chitinases from mycoparasitic fungi, such as Aphanocladium album and Trichoderma harzianum, but it had no potential secretory signal sequence in its amino terminus. Northern blot analysis showed that chi3 was transcribed during hyphal growth. These results suggest that chitinase III may function during morphogenesis in R. oligosporus.

Amino Acid Sequence↗