Selective inhibition of extracellular signal-regulated kinase (ERK) represents a potential approach for the treatment of cancer and other diseases; however, no selective inhibitors are currently ...available. Here, we describe an ERK-selective inhibitor, FR180204, and determine the structural basis of its selectivity. FR180204 inhibited the kinase activity of ERK1 and ERK2, with
K
i values 0.31 and 0.14
μM, respectively. Lineweaver–Burk analysis of the binding interaction revealed that FR180204 acted as competitive inhibitor of ATP. In mink lung epithelial Mv1Lu cells, FR180204 inhibited TGFβ-induced luciferase-expression. X-ray crystal structure analysis of the human ERK2/FR180204 complex revealed that Q105, D106, L156, and C166, which form the ATP-binding pocket on ERK, play important roles in the drug/protein interaction. These results suggest that FR180204 is an ERK-selective and cell-permeable inhibitor, and could be useful for elucidating the roles of ERK as well as for drug development.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UL, UM, UPCLJ, UPUK
RATIONALE:It is well established that the migration and proliferation of vascular smooth muscle cells (VSMCs) have major roles in the vascular remodeling process. Our previous study showed that the ...Akt substrate Girdin, which is expressed in VSMCs and endothelial cells, is essential for postnatal angiogenesis. However, the function of Girdin and its Akt-mediated phosphorylation in VSMCs and their in vivo roles in vascular remodeling remain to be elucidated.
OBJECTIVE:We investigated the function of Girdin and its Akt-mediated phosphorylation using cultured VSMCs and animal models of vascular remodeling.
METHODS AND RESULTS:The depletion of Girdin by RNA interference disrupted the rearrangement of the actin cytoskeleton in VSMCs, resulting in impaired cell migration. The depletion of Girdin also inhibited VSMC proliferation. Girdin expression was highly upregulated and its serine at position 1416 was phosphorylated in the neointima of carotid arteries after balloon injury in a rat model. The introduction of an adenovirus harboring short hairpin RNA against Girdin attenuated the proliferation of VSMCs and neointima formation without affecting reendothelialization. Furthermore, we found that neointima formation after femoral wire injury was significantly attenuated in Girdin S1416A knock-in mice, in which the Akt phosphorylation site of Girdin was mutated, thus indicating a major role for Girdin phosphorylation in vascular remodeling.
CONCLUSIONS:These findings indicate that Girdin and its Akt-mediated phosphorylation have major roles in the migration and proliferation of VSMCs and vascular remodeling, making the Akt/Girdin signaling pathway a potential target for the development of new therapeutics for vascular diseases.
The localization of salt-induced H
2
O
2
accumulation in the leaves of rice was examined using 3,3-diaminobenzidine and CeCl
3
staining at ultrastructure level. When the 3-week-old rice plants were ...affected by 100 mM NaCl for 14 days, the swelling of thylakoids and the destruction of thylakoid membranes were observed. H
2
O
2
accumulation was also observed in the chloroplast of the leaf treated with NaCl. The electron dense products of 3,3-diaminobenzidine and CeCl
3
were mainly observed especially around the swelling of thylakoids. H
2
O
2
accumulation and any ultrastructural changes were not observed in the chloroplasts under dark condition. Furthermore, treatment with ascorbic acid suppressed both H
2
O
2
accumulation and the changes in chloroplast ultrastructure. These results suggest that light-induced production of excess H
2
O
2
under salinity is responsible for the changes in chloroplast ultrastructure. H
2
O
2
accumulation was also observed in the mitochondria, peroxisomes, plasma membrane, and cell walls under light but not dark, suggesting that these organelles are also the source of H
2
O
2
and the production is light dependent under salinity.
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EMUNI, FIS, FZAB, GEOZS, GIS, IJS, IMTLJ, KILJ, KISLJ, MFDPS, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, SBMB, SBNM, UKNU, UL, UM, UPUK, VKSCE, VSZLJ, ZAGLJ
Cnidarians are considered ancestral metazoans and, therefore, are important taxa for studying animal evolution. However, little is known about the group’s genome size (
C
value), which is an ...important parameter in whole-genome sequencing. To address this issue, we measured the
C
values of 27 cnidarian species from Japan, using flow cytometry, and found that they ranged from 0.26 to 3.56 pg. Excluding the results for
Agalma elegans
and
Physalia physalis
(order Siphonophorae), which had the highest
C
values among the species included in the present study, the
C
values for the cnidarians were 0.26–1.49 pg. In particular, we found that hydrozoans possessed relatively large and wide-ranging
C
values, indicating that evolution within the group involved considerable gains or losses of genomic content. Overall, the
C
values reported in the present study could be valuable for whole-genome sequencing, using next-generation sequencers, and for future research in cytogenetics.
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CEKLJ, DOBA, EMUNI, FIS, FZAB, GEOZS, GIS, IJS, IMTLJ, IZUM, KILJ, KISLJ, MFDPS, NLZOH, NUK, OBVAL, OILJ, PILJ, PNG, SAZU, SBCE, SBJE, SBMB, SBNM, UILJ, UKNU, UL, UM, UPUK, VKSCE, ZAGLJ
► Conformal electroless barrier and seed layers were formed in high aspect ratio TSV. ► Au nanoparticles of 20
nm diameter were used as a catalyst. ► Adhesion strength of the diffusion barrier films ...are enhanced with annealing at 300
°C. ► This technology enables all-wet fabrication of Cu-filled TSV.
An all-wet process was achieved using electroless deposition of barrier and Cu seed layers for fabrication of a high aspect ratio through-Si via (TSV). Formation of a thin barrier metal layer of Ni–B, Co–B and Co–W–B is possible using a Au nanoparticle (AuNP) catalyst, which is densely adsorbed on the SiO
2 of the TSV sidewall. A silane coupling agent of 3-aminopropyl-triethoxysilane is effective for enhancement of the density of adsorption for AuNP. A conformal electroless Cu layer is deposited on the barrier layer by displacement plating without a catalyst. The adhesion strength between the electroless barrier layer and the SiO
2 substrate is increased by annealing at 300
°C. These results strongly suggest that an all-wet process for the formation of Cu-filled TSV with a high aspect ratio is practically possible.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UL, UM, UPCLJ, UPUK
A new family, genus and species of cubozoan box jellyfish belonging to the order Chirodropida is reported from the eastern Japan. Meteorona kishinouyei gen. et sp. n. possesses the following unique ...morphological characters with respect to other known species in the Chirodropida: having one tentacle per scalpel-like unbranched pedalium and slightly raised unbranched gastric saccules. A comparative table of the primary diagnostic characters of genus and order in the Chirodropida is given. The order Chirodropida is redefined. The family Chiropsellidae is established. Discussion is provided on the implications for these findings on our current understanding of Cubozoan systematics.
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IZUM, KILJ, NUK, PILJ, PNG, SAZU, UL, UM, UPUK
The accumulation of glycinebetaine (GB) is one of the adaptive strategies to adverse salt stress conditions. Although it has been demonstrated that barley plants accumulate GB in response to salt ...stress and various studies focused on GB synthesis were performed, its transport mechanism is still unclear. In this study, we identified a novel gene, HvProT2, encoding Hordeum vulgare GB/proline transporter from barley plants. Heterologous expression in yeast (Saccharomyces cerevisiae) mutant demonstrated that the affinity of HvProT2 was highest for GB, intermediate for proline and lowest for γ-aminobutyric acid. Transient expression of fusions of HvProT2 and green fluorescent protein in onion epidermal cells revealed that HvProT2 is localized at the plasma membrane. Relative quantification of mRNA level of HvProT2 using semi-quantitative reverse transcription-polymerase chain reaction analysis showed that HvProT2 is constitutively expressed in both leaves and roots, and the expression level was higher in old leaves than young leaves and roots. Moreover, we found that HvProT2 was expressed in the mestome sheath and lateral root cap cells. We discussed the possible involvement of HvProT2 for salt stress tolerance.
SUMMARY
Both sexes of the Japanese giant box jellyfish Morbakka virulenta were collected from the Seto Inland Sea, western Japan in December 2011, in order to observe the developmental processes from ...polyps to medusae. The medusa production in M. virulenta is up to now a unique process in cubozoans in that it exhibits a form of monodisc strobilation where the polyp is regenerated before the medusa detaches. This mode of medusa production was previously thought to be exclusive to scyphozoans. The general shape of young medusae resembles that of other cubozoans such as Alatina moseri and Copula sivickisi, but is differentiated from these by the short capitate tentacles and the lack of gastric filaments in the stomach. The unique medusa production of M. virulenta highly implies a phylogenetic similarity between cubozoans and scyphozoans.
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DOBA, FZAB, GIS, IJS, IZUM, KILJ, NLZOH, NUK, OILJ, PILJ, PNG, SAZU, SBCE, SBMB, UILJ, UKNU, UL, UM, UPUK
This paper studied the enacting process of the Application fee for Building confirmation. Conclusion of this study is as follows; 1) In the process of enacting the Building Standards Law, the maximum ...amount and the maximum amount above a certain scale were added to the article after the legal examination. 2) In the process of enacting the Enforcement Order, detailed provisions were established with eight levels of area classification. 3) In order to make the maximum upper limit equal to the conventional amount, the area scale of the corresponding category was set too small as a standard for the future.
Kinases of mitogen-activated protein kinase (MAPK) cascades, including extracellular signal-regulated protein kinase (ERK), represent likely targets for pharmacological intervention in proliferative ...diseases. Here, we report that FR148083 inhibits ERK2 enzyme activity and TGFβ-induced AP-1-dependent luciferase expression with respective IC
50 values of 0.08 and 0.05
μM. FR265083 (1′–2′ dihydro form) and FR263574 (1′–2′ and 7′–8′ tetrahydro form) exhibited 5.5-fold less and no activity, respectively, indicating that both the α,β-unsaturated ketone and the conformation of the lactone ring contribute to this inhibitory activity. The X-ray crystal structure of the ERK2/FR148083 complex revealed that the compound binds to the ATP binding site of ERK2, involving a covalent bond to Sγ of ERK2 Cys166, hydrogen bonds with the backbone NH of Met108, Nζ of Lys114, backbone C=O of Ser153, Nδ2 of Asn154, and hydrophobic interactions with the side chains of Ile31, Val39, Ala52, and Leu156. The covalent bond motif in the ERK2/FR148083 complex assures that the inhibitor has high activity for ERK2 and no activity for other MAPKs such as JNK1 and p38MAPKα/β/γ/δ which have leucine residues at the site corresponding to Cys166 in ERK2. On the other hand, MEK1 and MKK7, kinases of the MAPKK family which also can be inhibited by FR148083, contain a cysteine residue corresponding to Cys166 of ERK2. The covalent binding to the common cysteine residue in the ATP-binding site is therefore likely to play a crucial role in the inhibitory activity for these MAP kinases. These findings on the molecular recognition mechanisms of FR148083 for kinases with Cys166 should provide a novel strategy for the pharmacological intervention of MAPK cascades.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UL, UM, UPCLJ, UPUK
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