ALBERT

Description: Vacuolar H + -ATPase (V-ATPase) is responsible for the acidification of eukaryotic intracellular compartments and plays an important role in oxidative stress response (OSR), but its molecular bases are largely unknown. Here, we investigated how V-ATPase is involved in the OSR by using a strain lacking VPH2 , which encodes an assembly factor of V-ATPase, in the pathogenic fungus Candida glabrata . The loss of Vph2 resulted in increased H 2 O 2 sensitivity and intracellular reactive oxygen species (ROS) level independently of mitochondrial functions. The vph2 mutant also displayed growth defects under alkaline conditions accompanied by the accumulation of intracellular ROS and these phenotypes were recovered in the presence of the ROS scavenger N-acetyl- l -cysteine. Both expression and activity levels of mitochondrial manganese superoxide dismutase (Sod2) and catalase (Cta1) were decreased in the vph2 mutant. Phenotypic analyses of strains lacking and overexpressing these genes revealed that Sod2 and Cta1 play a predominant role in endogenous and exogenous OSR, respectively. Furthermore, supplementation of copper and iron restored the expression of SOD2 specifically in the vph2 mutant, suggesting that the homeostasis of intracellular cupper and iron levels maintained by V-ATPase was important for the Sod2-mediated OSR. This report demonstrates novel roles of V-ATPase in the OSR in C. glabrata .

Description: N -glycosylation has been shown to be important for the stability of some glycoproteins. Isopullulanase (IPU), a polysaccharide-hydrolyzing enzyme, is a highly N -glycosylated protein, and IPU deglycosylation results in a decrease in thermostability. To investigate the function of N -glycan in IPU, we focused on an N -glycosylated residue located in the vicinity of the active site, Asn448. The thermostabilities of three IPU variants, Y440A, N448A and S450A, were 0.5–8.4°C lower than the wild-type enzyme. The crystal structure of endoglycosidase H (Endo H)-treated N448A variant was determined. There are four IPU molecules, Mol-A, B, C and D, in the asymmetric unit. The conformation of a loop composed of amino acid residues 435–455 in Mol-C was identical to wild-type IPU, whereas the conformations of this loop in Mol-A, Mol-B and Mol-D were different from each other. These results suggest that the Asn448 side chain is primarily important for the stability of IPU. Our results indicate that mutation of only N -glycosylated Asn residue may lead to incorrect conclusion for the evaluation of the function of N -glycan. Usually, the structures of N -glycosylation sites form an extended configuration in IPU; however, the Asn448 site had an atypical structure that lacked this configuration.

Description: Pagan is one of the largest volcanoes along the Mariana arc volcanic front. It has a maximum elevation of 570 m (Mt. Pagan), but its submarine flanks descend to 2000–3000 m below sea level, and are unexplored. Bathymetric mapping and ROV Hyper-Dolphin dives (HPD1147 and HPD1148) on the submarine NE and SW flanks of Pagan were carried out during cruise NT10-12 of R.V. Natsushima in July 2010. There are no systematic compositional differences between subaerial lavas reported in the literature and differentiated submarine lavas collected in HPD1148, with 〈7 wt % MgO, suggesting they are derived from the same magmatic system. However, these differentiated lavas show complexities including magma mixing; thus we concentrate on magnesian submarine lavas (〉7 wt % MgO). Twenty least-fractionated basalts (48·5–50 wt % SiO 2 ) collected during HPD1147 extend to higher MgO (10–11 wt %) and Mg# (66–70) than the subaerial lavas. Olivine (up to Fo 94 ) and spinel (Cr# up to 0·8) compositions suggest that these Pagan primitive magmas formed from high degrees of mantle melting. Two basalt types can be distinguished based on their geochemistry at similar (10–11 wt %) MgO; these erupted recently, 500 m apart. Both contain clinopyroxene and olivine phenocrysts and are referred to as COB1 and COB2. Lower TiO 2 , FeO, Na 2 O, K 2 O, incompatible trace element abundances, and Nb/Yb suggest that COB1 formed from higher degrees of mantle melting. In addition, light rare earth element (LREE) enrichment and higher Th/Nb in COB2 contrast with LREE depletion and lower Th/Nb in COB1. Higher Ba/Th and Ba/Nb and lower Th/Nb indicate that the main subduction addition in COB1 was dominated by hydrous fluid, whereas that in COB2 was dominated by sediment melt. Sr–Nd–Pb–Hf isotopes are also consistent with this interpretation. These observations suggest that the subduction component responsible for the greater degree of melting of the COB1 source was mostly hydrous fluid. The origin of such different metasomatic agents resulted in different primary magmas forming in the same volcano. Both hydrous fluid and sediment melt components may have unmixed from an originally homogeneous supercritical fluid in or above the subducting slab below the volcanic front. These may have been added separately to the mantle wedge peridotite (mantle diapir) and resulted in two neighboring but completely different primary magmas from the same diapir. Moreover, these primitive lavas suggest that even for intra-oceanic arcs assimilation–fractional crystallization is inevitable when these magmas evolve in the crust and, in addition, that phlogopite is present in their mantle residue and thus played an important role in their genesis.

Description: Major and trace element and Sr–Nd–Pb isotope data for whole-rocks and major element data for minerals within basalt samples from the Chugaryeong volcano, an intra-plate back-arc volcanic centre in the central part of the Korean Peninsula, are used to address the process of magma genesis in the deep back-arc region of eastern Asia. There are two lava flow units at Chugaryeong volcano: the Chongok (0·50 Ma) and the Chatan (0·15 Ma) basalts. These basalts have similar MgO (9·1–10·4 wt %) but exhibit differences in their major and trace element and isotope compositions. The Chongok basalt has higher TiO 2 , Al 2 O 3 , Na 2 O, K 2 O, P 2 O 5 , Cr 2 O 3 , large ion lithophile elements (LILE), high field strength elements (HFSE), and rare earth elements (REE), and lower FeO*, SiO 2 , and CaO than the Chatan basalt. In addition, the Chongok basalt has more radiogenic 143 Nd/ 144 Nd and 206 Pb/ 204 Pb, and less radiogenic 87 Sr/ 86 Sr and 208 Pb/ 204 Pb than the Chatan basalt. Chi-square tests for the major elements indicate that crystal fractionation can explain the chemical variations within each basalt suite; intra-crustal processes, including crystal fractionation and assimilation of continental crust, cannot result in the formation of one basalt suite from the other. The Sr–Nd–Pb isotopic compositions of the Chongok and Chatan basalts plot on mixing hyperbolae between peridotite mantle xenoliths from the area and a fluid flux derived from a mixture of ancient and recent sediments. The trace element compositions of the estimated primary melts for the two basalt suites suggest different degrees of partial melting of a common enriched mantle source that was metasomatized by a Ba-, K-, Pb-, and Sr-rich fluid. The estimated degree of melting increased with time from ~7·5% for the Chongok basalt to ~10% for the Chatan basalt. The source mantle for the Chatan basalt is more enriched in Ba and Pb, indicating a greater fluid flux than for the Chongok basalt. This suggests that melting of the source mantle increased with time, sustained by an increased sediment-derived fluid flux from the deeper upper mantle.

Description: Initial subduction-related boninitic magmatism occurred between 48 and 44 Ma in the Izu–Bonin–Mariana (IBM) arc. High-Mg adakites and low-Ca boninites have been dredged from the Bonin Ridge fore-arc seamount. Whole-rock 40 Ar/ 39 Ar ages suggest that the boninite (44·0 ± 1·4 Ma) and adakite (43·1 ± 1·0 and 40·8 ± 0·8 Ma) magmatism overlapped, or that the adakite magmatism occurred slightly later than the boninite magmatism. The low-Ca boninites are high-Mg andesites and exhibit U-shaped rare earth element (REE) patterns with an elevated average Mg# of 0·78 [Mg# = Mg/(Mg + Fe) molar ratio] and Ni content of 667 ppm. The high-Mg adakites are andesitic to dacitic in composition; they exhibit markedly high Sr contents and low Y contents and are highly enriched in light REE but depleted in heavy REE, with an average Mg# of 0·79 and Ni content of 433 ppm. A geochemical mass-balance model (Arc Basalt Simulator Version 3) indicates that both magma types could be generated by partial melting of a depleted mantle source fluxed by water-rich slab-derived melts in a hot subduction environment, comparable with the present-day South Chile (ridge subduction) or Southwest Japan (young slab subduction) arcs. An extremely high slab melt flux of 22% is required for the formation of the high-Mg adakite, whereas a low flux of 3% is sufficient for the low-Ca boninite. The low-Ca boninite requires a high-temperature shallow slab (854°C, 2·7 GPa on average), consisting of altered oceanic crust of the Pacific plate and volcaniclastic sediments from HIMU seamounts, and high-temperature shallow mantle melting (1216°C, 0·8 GPa) of depleted Indian mid-ocean ridge basalt (MORB)-type mantle. These modelled conditions are consistent with the occurrence of hot shallow mantle wedge melting in the initial subduction zone at the boundary between Pacific- and Indian-type mantle domains, as suggested by previous studies. In contrast, high-Mg adakite requires a higher temperature and deeper slab (929°C, 4·1 GPa), with the same slab components and slightly deeper but less hot melting (1130°C, 1·1 GPa) of HIMU-type depleted mantle, to satisfy the low Hf isotope ratios. This may occur because of the subsequent cooling of the mantle wedge by the establishment of the subduction system after the boninite magmatism and involvement of a small volume of an isotopically enriched mantle source embedded in the Indian-type mantle. The petrogenetic conditions provide constraints for reconstructing the tectonic settings of the early IBM arc. The hot subduction model would be consistent with the tectonic models with regard to the initiation of subduction associated with fore-arc spreading; this allowed the upwelling of the asthenospheric mantle to generate slab melts from the old Pacific plate slab and hot shallow mantle melting by slab melt fluxing for both boninite and adakite activities.

Description: Mutations of Filamin genes, which encode actin-binding proteins, cause a wide range of congenital developmental malformations in humans, mainly skeletal abnormalities. However, the molecular mechanisms underlying Filamin functions in skeletal system formation remain elusive. In our screen to identify skeletal development molecules, we found that Cfm ( Fam101 ) genes, Cfm1 ( Fam101b ) and Cfm2 ( Fam101a ), are predominantly co-expressed in developing cartilage and intervertebral discs (IVDs). To investigate the functional role of Cfm genes in skeletal development, we generated single knockout mice for Cfm1 and Cfm2 , as well as Cfm1/Cfm2 double-knockout ( Cfm DKO) mice, by targeted gene disruption. Mice with loss of a single Cfm gene displayed no overt phenotype, whereas Cfm DKO mice showed skeletal malformations including spinal curvatures, vertebral fusions and impairment of bone growth, showing that the phenotypes of Cfm DKO mice resemble those of Filamin B (Flnb) -deficient mice. The number of cartilaginous cells in IVDs is remarkably reduced, and chondrocytes are moderately reduced in Cfm DKO mice. We observed increased apoptosis and decreased proliferation in Cfm DKO cartilaginous cells. In addition to direct interaction between Cfm and Filamin proteins in developing chondrocytes, we showed that Cfm is required for the interaction between Flnb and Smad3, which was reported to regulate Runx2 expression. Furthermore, we found that Cfm DKO primary chondrocytes showed decreased cellular size and fewer actin bundles compared with those of wild-type chondrocytes. These results suggest that Cfms are essential partner molecules of Flnb in regulating differentiation and proliferation of chondryocytes and actin dynamics.

Description: It has long been thought that the low-K tholeiitic (TH) and medium-K calc-alkaline (CA) lavas in the NE Japan arc were produced by fractional crystallization from mantle-derived basalt magmas, and that the latter formed from mixing of mafic and felsic magmas, both derived from a common primary TH basalt through fractionation. An alternative view was recently proposed on the basis of Sr isotope microanalysis of plagioclase phenocrysts from the Zao volcano, suggesting that (1) the low-K TH basaltic andesites formed by melting of lower crustal amphibolite and that (2) the medium-K CA basalts to andesites formed by mixing of mantle-derived basalt and crustal TH melts. To investigate further the origin of the ‘primary’ low-K TH and medium-K CA basalts, we investigated basalts and andesites from Azuma volcano. Azuma is a Quaternary eruption center at the volcanic front in the NE Japan arc that has erupted two types of basalt: (1) radiogenic-Sr ( 87 Sr/ 86 Sr = 0·7058–0·7062) low-K TH basalt lavas without evidence of magma mixing and assimilation; (2) unradiogenic-Sr ( 87 Sr/ 86 Sr = 0·7039–0·7041) medium-K CA basalt lavas with subtle evidence for magma mixing. Associated intermediate lavas are voluminous and are all (3) mildly radiogenic-Sr ( 87 Sr/ 86 Sr = 0·7044–0·7055) medium-K andesites, all of which have CA affinities with evidence for rigorous magma mixing but no crustal assimilation. The low-K TH basalt has an isotopic composition similar to that of crustal granitoids beneath Azuma and has a composition indicating that it potentially formed from a high-degree lower crustal amphibolite melt. The medium-K CA basalt has a basaltic groundmass with Mg-rich olivine (Fo 89 ) and calcic plagioclase phenocrysts (An 90 ) and the most unradiogenic Sr ( 87 Sr/ 86 Sr = 0·7037–0·7038), suggesting that it originated from a primary mantle melt. Major and trace element microanalysis of the basaltic groundmass indicates that the primary magma composition is close to high-K. We conclude that the mantle-derived basalt at Azuma is the result of a high- to medium-K magma that was later mixed with a low-K TH basalt melt from the amphibolitic lower crust to form medium-K CA basalts and andesites. This supports the view of a lower crustal origin of the low-K TH basalts and simultaneously requires a reappraisal of the origin of the across-arc variation in K contents of the mantle-derived primary arc basalts, as the high- to medium-K CA basalt is geochemically fairly similar to the high-K rear-arc basalt in the NE Japan arc.

Description: In this study, we have investigated the specific binding proteins of Zinc- l -carnosine (Polaprezinc) using Polaprezinc-affinity column chromatography in vitro . A protein having a 70-kDa molecular mass was eluted by the linear gradient of 0 – 1.0 mM Polaprezinc from the affinity column and the protein was identified as the molecular chaperone HSP70 by immunoblotting. The chaperone activity of HSP70 was completely suppressed by Polaprezinc. The ATPase activity of HSP70 was affected to some extent by the reagent. In the circular dichroism (CD) spectrum, the secondary structure of HSP70 was changed in the presence of Polaprezinc, i.e. it decreased in the α-helix. We have determined the Polaprezinc-binding domain of HSP70 by using recombinant HSP70N- and C-domains. Although Polaprezinc could bind to both the N-terminal and the C-terminal of HSP70, the HSP70N-domain has a high affinity to the drug. Regarding the peptide cleavage of the HSP70N- and C-domains with proteinase K, the intact HSP70N still remained in the presence of Polaprezinc. On the other hand, the quantity of the intact C-domain slightly decreased under the same conditions along with the newly digested small peptides appeared. It has been suggested that Polaprezinc binds to HSP70 especially in the N-domains, suppresses the chaperone activity and delays an ATPase activities of HSP70.

Description: We conducted compressional, tensile, and torsional creep experiments on fine-grained forsterite plus Ca-bearing pyroxene aggregates. A distinct microstructure with aggregation of the same phase in the direction of compression was formed in our samples after all the experiments. The stress–strain rate relationship, grain-size dependent flow strength, and the achievement of large tensile strain all indicate that samples underwent creep due to grain boundary sliding (GBS). As a result of GBS, grain-switching events allow dispersed phases to contact grains of the same phase and orient in the direction of compression. We identify similar aggregated microstructures in previously reported micrographs of polymineralic granite-origin ultramylonites. Mineral phase mixing through GBS, which helps to retain fine grain size in rocks due to grain boundary pinning, has been speculated to occur during formation of mylonites. However, our results contradict this hypothesis because mineral aggregation through GBS promotes demixing rather than mixing of the mineral phases. GBS processes alone will not promote a transformation of well-developed monomineralic bands to polymineralic bands during mylonitization.

Description: Vasohibin-1 (VASH1) is an angiogenesis inhibitor synthesized by endothelial cells (ECs) under conditions associated with physiological and pathological angiogenesis including cancers. VASH1, which is a 44-kDa protein, is processed after its translation and secretion, and a 29 kDa product cleaved both N-terminal and C-terminal end loses its anti-angiogenic activity. Here, we tested whether cancer cells modulate the processing of VASH1. When mouse EC line MS1 stably overexpressing the human VASH1 gene (MS1-hVASH1) and various cancer cell lines were co-cultured, there was an increased processing of hVASH1 protein in the culture media. This augmented processing was abrogated by a general cysteine protease inhibitor, E-64, and also by a specific calpain inhibitor, MDL28170. Recombinant hVASH1 protein was degraded by µ-calpain in vitro , which degradation was blocked by calpeptin. Conditioned media from co-cultures had little effect on the migration of human umbilical vein endothelial cells, but exhibited an inhibitory effect on their migration when collected in the presence of MDL28170; and this inhibitory effect was blocked by neutralizing anti-hVASH1 mAb. These results indicate that cancer cells proteolytically inactivate VASH1 protein secreted by ECs in the tumour microenvironment.