Fracture-induced amorphization of polycrystalline SiO2 stishovite: a potential platform for toughening in ceramics.

Silicon dioxide has eight stable crystalline phases at conditions of the Earth's rocky parts. Many metastable phases including amorphous phases have been known, which indicates the presence of large kinetic barriers. As a consequence, some crystalline silica phases transform to amorphous phases by bypassing the liquid via two different pathways. Here we show a new pathway, a fracture-induced amorphization of stishovite that is a high-pressure polymorph. The amorphization accompanies a huge volume expansion of ~100% and occurs in a thin layer whose thickness from the fracture surface is several tens of nanometers. Amorphous silica materials that look like strings or worms were observed on the fracture surfaces. The amount of amorphous silica near the fracture surfaces is positively correlated with indentation fracture toughness. This result indicates that the fracture-induced amorphization causes toughening of stishovite polycrystals. The fracture-induced solid-state amorphization may provide a potential platform for toughening in ceramics.

The endosomal Na(+)/H(+) exchanger contributes to multivesicular body formation by regulating the recruitment of ESCRT-0 Vps27p to the endosomal membrane.

Multivesicular bodies (MVBs) are late endosomal compartments containing luminal vesicles (MVB vesicles) that are formed by inward budding of the endosomal membrane. In budding yeast, MVBs are an important cellular mechanism for the transport of membrane proteins to the vacuolar lumen. This process requires a class E subset of vacuolar protein sorting (VPS) genes. VPS44 (allelic to NHX1) encodes an endosome-localized Na(+)/H(+) exchanger. The function of the VPS44 exchanger in the context of vacuolar protein transport is largely unknown. Using a cell-free MVB formation assay system, we demonstrated that Nhx1p is required for the efficient formation of MVB vesicles in the late endosome. The recruitment of Vps27p, a class E Vps protein, to the endosomal membrane was dependent on Nhx1p activity and was enhanced by an acidic pH at the endosomal surface. Taken together, we propose that Nhx1p contributes to MVB formation by the recruitment of Vps27p to the endosomal membrane, possibly through Nhx1p antiporter activity.

Na+/H+ exchanger isoform 6 (NHE6/SLC9A6) is involved in clathrin-dependent endocytosis of transferrin.

In mammalian cells, nine conserved isoforms of the Na(+)/H(+) exchanger (NHE) are known to be important for pH regulation of the cytoplasm and organellar lumens. NHE1-5 are localized to the plasma membrane, whereas NHE6-9 are localized to distinct organelles. NHE6 is localized predominantly in endosomal compartments but is also found in the plasma membrane. To investigate the role of NHE6 in endocytosis, we established NHE6-knockdown HeLa cells and analyzed the effect of this knockdown on endocytotic events. The expression level of NHE6 in knockdown cells was decreased to ∼15% of the level seen in control cells. Uptake of transferrin was also decreased. No effect was found on the endocytosis of epidermal growth factor or on the cholera toxin B subunit. Moreover, in the NHE6-knockdown cells, transferrin uptake was found to be affected in the early stages of endocytosis. Microscopic analysis revealed that, at 2 min after the onset of endocytosis, colocalization of NHE6, clathrin, and transferrin was observed, which suggests that NHE6 was localized to endocytotic, clathrin-coated vesicles. In addition, in knockdown cells, transferrin-positive endosomes were acidified, but no effect was found on cytoplasmic pH. In cells overexpressing wild-type NHE6, increased transferrin uptake was observed, but no such increase was seen in cells overexpressing mutant NHE6 deficient in ion transport. The luminal pH in transferrin-positive endosomes was alkalized in cells overexpressing wild-type NHE6 but normal in cells overexpressing mutant NHE6. These observations suggest that NHE6 regulates clathrin-dependent endocytosis of transferrin via pH regulation.

A loss-of-function mutation in the SLC9A6 gene causes X-linked mental retardation resembling Angelman syndrome.

SLC9A6 mutations have been reported in families in whom X-linked mental retardation (XMR) mimics Angelman syndrome (AS). However, the relative importance of SLC9A6 mutations in patients with an AS-like phenotype or XMR has not been fully investigated. Here, the involvement of SLC9A6 mutations in 22 males initially suspected to have AS but found on genetic testing not to have AS (AS-like cohort), and 104 male patients with XMR (XMR cohort), was investigated. A novel SLC9A6 mutation (c.441delG, p.S147fs) was identified in one patient in the AS-like cohort, but no mutation was identified in XMR cohort, suggesting mutations in SLC9A6 are not a major cause of the AS-like phenotype or XMR. The patient with the SLC9A6 mutation showed the typical AS phenotype, further demonstrating the similarity between patients with AS and those with SLC9A6 mutations. To clarify the effect of the SLC9A6 mutation, we performed RT-PCR and Western blot analysis on lymphoblastoid cells from the patient. Expression of the mutated transcript was significantly reduced, but was restored by cycloheximide treatment, indicating the presence of nonsense mediated mRNA decay. Western blot analysis demonstrated absence of the normal NHE6 protein encoded for by SLC9A6. Taken together, these findings indicate a loss-of-function mutation in SLC9A6 caused the phenotype in our patient.

Evidence that "brain-specific" FOX-1, FOX-2, and nPTB alternatively spliced isoforms are produced in the lens.

PURPOSE: Alternative RNA splicing is essential in development and more rapid physiological processes that include disease mechanisms. Studies over the last 20 years demonstrated that RNA binding protein families, which mediate the alternative splicing of a large percentage of genes in mammals, contain isoforms with mutually exclusive expression in non-neural and neural progenitor cells vs. post-mitotic neurons, and regulate the comprehensive reprogramming of alternative splicing during neurogenesis. Polypyrimidine tract binding (PTB) proteins and Fox-1 proteins also undergo mutually exclusive alternative splicing in neural and non-neural cells that regulates their tissue-specific expression and splicing activities. Over the past 50 years, striking morphological similarities noted between lens fiber cells and neurons suggested that cell biology processes and gene expression profiles may be shared as well. Here, we examined mouse and rat lenses to determine if alternative splicing of neuronal nPTB and Fox-1/Fox-2 isoforms also occurs in lenses. METHODS: Immunoblot, immunofluorescence, and RT-PCR were used to examine expression and alternative splicing of transcripts in lens and brain. RESULTS: We demonstrated that exon 10 is predominantly included in nPTB transcripts consistent with nPTB protein in lenses, and that alternatively spliced Fox-1/-2 lens transcripts contain exons that have been considered neuron-specific. We identified a 3' alternative Fox-1 exon in lenses that encodes a nuclear localization signal consistent with its protein distribution detected in fiber cells. Neuronal alternative splicing of kinesin KIF1Bß2 has been associated with PTB/nPTB and Fox-2, and we found that two 'neuron-specific' exons are also included in lenses. CONCLUSIONS: The present study provides evidence that alternative neuronal nPTB and Fox-1/Fox-2 isoforms are also produced in lenses. These findings raise questions regarding the extent these factors contribute to a similar reprogramming of alternative splicing during lens differentiation, and the degree that alternative gene transcripts produced during neurogenesis are also expressed in the lens.

Dual functional significance of calcineurin homologous protein 1 binding to Na(+)/H(+) exchanger isoform 1.

Calcineurin homologous protein 1 (CHP1) binds to the hydrophilic tail of the Na(+)/H(+) exchanger isoform 1 (NHE1). Previous gene knockout of CHP1 revealed that the loss of CHP1 caused a decrease in the total amount of NHE1, suggesting the destabilization of NHE1 molecules without CHP1 (Matsushita et al., Am J Physiol Cell Physiol 293: C246-C254, 2007). However, Pang et al. (J Biol Chem 276: 17367-17372, 2001) reported that NHE1 without a CHP1 binding site was found in the plasma membrane, suggesting no requirement of CHP1 binding for plasma membrane localization of NHE1. Here, the functional significance of CHP1 binding to NHE1 was examined to resolve these contradictory results. In CV1 cells, which overexpressed wild-type NHE1, overexpression of CHP1 caused an increase in both the total amount of NHE1 and the colocalization of NHE1 and CHP1 at the plasma membrane. This provided new visual evidence of the localization of NHE1 from endoplasmic reticulum to the plasma membrane upon CHP1 binding. An immunoprecipitation assay showed that the expression of CHP1 reduced the ubiquitination of NHE1 and/or its associated proteins. Mutant NHE1s without CHP1 binding site exhibited a modest localization to the plasma membrane. After reaching the plasma membrane, these mutant NHE1s exhibited shorter half-lives than the wild-type NHE1 with CHP1. The results suggest a dual functional significance of CHP1 and its binding region: 1) binding of CHP1 stabilizes NHE1 and increases its plasma membrane localization by masking a NHE1 disposal signal, and 2) CHP1 binding is required for the antiporter activity.

Organellar Na+/H+ exchangers: novel players in organelle pH regulation and their emerging functions.

Mammalian Na+/H+ exchangers (NHEs) play a fundamental role in cellular ion homeostasis. NHEs exhibit an appreciable variation in expression, regulation, and physiological function, dictated by their dynamics in subcellular localization and/or interaction with regulatory proteins. In recent years, a subgroup of NHEs consisting of four isoforms has been identified, and its members predominantly localize to the membranes of the Golgi apparatus and endosomes. These organellar NHEs constitute a family of transporters with an emerging function in the regulation of luminal pH and in intracellular membrane trafficking as expressed, for example, in cell polarity development. Moreover, specific roles of a variety of cofactors, regulating the intracellular dynamics of these transporters, are also becoming apparent, thereby providing further insight into their mechanism of action and overall functioning. Interestingly, organellar NHEs have been related to mental disorders, implying a potential role in the brain, thus expanding the physiological significance of these transporters.

Boronization and Carburization of Superplastic Stainless Steel and Titanium-Based Alloys.

Bronization and carburization of fine-grain superplastic stainless steel is reviewed, and new experimental results for fine grain Ti88.5Al4.5V3Fe2Mo2 are reported. In superplastic duplex stainless steel, the diffusion of carbon and boron is faster than in non-superplastic duplex stainless steel. Further, diffusion is activated by uniaxial compressive stress. Moreover, non-superplastic duplex stainless steel shows typical grain boundary diffusion; however, inner grain diffusion is confirmed in superplastic stainless steel. The presence of Fe and Cr carbides or borides is confirmed by X-ray diffraction, which indicates that the diffused carbon and boron react with the Fe and Cr in superplastic stainless steel. The Vickers hardness of the carburized and boronized layers is similar to that achieved with other surface treatments such as electro-deposition. Diffusion of boron into the superplastic Ti88.5Al4.5V3Fe2Mo2 alloy was investigated. The hardness of the surface exposed to boron powder can be increased by annealing above the superplastic temperature. However, the Vickers hardness is lower than that of Ti boride.

Saccharomyces cerevisiae glucose signalling regulator Mth1p regulates the organellar Na+/H+ exchanger Nhx1p.

Organelle-localized NHEs (Na+/H+ exchangers) are found in cells from yeast to humans and contribute to organellar pH regulation by exporting H+ from the lumen to the cytosol coupled to an H+ gradient established by vacuolar H+-ATPase. The mechanisms underlying the regulation of organellar NHEs are largely unknown. In the present study, a yeast two-hybrid assay identified Mth1p as a new binding protein for Nhx1p, an organellar NHE in Saccharomyces cerevisiae. It was shown by an in vitro pull-down assay that Mth1p bound to the hydrophilic C-terminal half of Nhx1p, especially to the central portion of this region. Mth1p is known to bind to the cytoplasmic domain of the glucose sensor Snf3p/Rgt2p and also functions as a negative transcriptional regulator. Mth1p was expressed in cells grown in a medium containing galactose, but was lost (possibly degraded) when cells were grown in medium containing glucose as the sole carbon source. Deletion of the MTH1 gene increased cell growth compared with the wild-type when cells were grown in a medium containing galactose and with hygromycin or at an acidic pH. This resistance to hygromycin or acidic conditions was not observed for cells grown with glucose as the sole carbon source. Gene knockout of NHX1 increased the sensitivity to hygromycin and acidic pH. The increased resistance to hygromycin was reproduced by truncation of the Mth1p-binding region in Nhx1p. These results implicate Mth1p as a novel regulator of Nhx1p that responds to specific extracellular carbon sources.

A membrane-proximal region in the C-terminal tail of NHE7 is required for its distribution in the trans-Golgi network, distinct from NHE6 localization at endosomes.

Mammalian Na(+)/H(+) exchanger (NHE) isoform NHE6 is localized in sorting/recycling endosomes, whereas NHE7 is localized in the trans-Golgi network (TGN) and mid-trans-Golgi stacks. The mechanism targeting each NHE to a specific organelle is largely unknown, although the targeting is thought to be important for pH control in the lumen of various organelles. NHE6 and NHE7 exhibit distinct localization despite conserved amino acid sequences. To specify the intramolecular region involved in the specific localization, we examined the intracellular localization of chimeric NHE6 and NHE7 constructs. NHEs are composed of an N-terminal transmembrane domain (TM) and a C-terminal hydrophilic tail domain (Ct). Exchange of the Ct between the isoforms suggested that the Ct is required for the specific localization. We further split the Ct into three regions, and chimeras with various combinations of these small regions indicated that the most membrane-proximal region among the three contributes to the specific localization. Mutant forms of NHE7 with sequential alanine substitutions in the most membrane-proximal region, between residues 530 and 589, showed that two regions (residues 553-559 and 563-568) are required for NHE7-like localization. However, NHE6 with alanine substitutions in the membrane-proximal region exhibited no apparent change in localization. These results suggest that two membrane proximal regions (residues 533-559 and 563-568) play an important role in targeting NHE7 to the TGN.

The Na+/H+ exchanger NHE6 in the endosomal recycling system is involved in the development of apical bile canalicular surface domains in HepG2 cells.

Polarized epithelial cells develop and maintain distinct apical and basolateral surface domains despite a continuous flux of membranes between these domains. The Na(+)/H(+)exchanger NHE6 localizes to endosomes but its function is unknown. Here, we demonstrate that polarized hepatoma HepG2 cells express an NHE6.1 variant that localizes to recycling endosomes and colocalizes with transcytosing bulk membrane lipids. NHE6.1 knockdown or overexpression decreases or increases recycling endosome pH, respectively, and inhibits the maintenance of apical, bile canalicular plasma membranes and, concomitantly, apical lumens. NHE6.1 knockdown or overexpression has little effect on the de novo biogenesis of apical surface domains. NHE6.1 knockdown does not inhibit basolateral-to-apical transcytosis of bulk membrane lipids, but it does promote their progressive loss from the apical surface, leaving cells unable to efficiently retain bulk membrane and bile canalicular proteins at the apical surface. The data suggest that a limited range of endosome pH mediated by NHE6.1 is important for securing the polarized distribution of membrane lipids at the apical surface and maintenance of apical bile canaliculi in HepG2 cells and hence cell polarity. This study underscores the emerging role of the endosomal recycling system in apical surface development and identifies NHE6 as a novel regulatory protein in this process.

Intermolecular cross-linking of monomers in Helicobacter pylori Na+/H+ antiporter NhaA at the dimer interface inhibits antiporter activity.

We have previously shown that HPNhaA (Helicobacter pylori Na+/H+ antiporter) forms an oligomer in a native membrane of Escherichia coli, and conformational changes of oligomer occur between monomers of the oligomer during ion transport. In the present study, we use Blue-native PAGE to show that HPNhaA forms a dimer. Cysteine-scanning mutagenesis of residues 55-61 in a putative beta-sheet region of loop1 and subsequent functional analyses revealed that the Q58C mutation resulted in an intermolecular disulfide bond. G56C, I59C and G60C were found to be cross-linked by bifunctional cross-linkers. Furthermore, the Q58E mutant did not form a dimer, possibly due to electrostatic repulsion between monomers. These results imply that Gln-58 and the flanking sequence in the putative beta-sheet of the monomer are located close to the identical residues in the dimer. The Q58C mutant of NhaA was almost inactive under non-reducing conditions, and activity was restored under reducing conditions. This result showed that cross-linking at the dimer interface reduces transporter activity by interfering with the flexible association between the monomers. A mutant HPNhaA protein with three amino acid substitutions at residues 57-59 did not form a dimer, and yet was active, indicating that the monomer is functional.

Altered motor activity of alternative splice variants of the mammalian kinesin-3 protein KIF1B.

Several mammalian kinesin motor proteins exist as multiple isoforms that arise from alternative splicing of a single gene. However, the roles of many motor protein splice variants remain unclear. The kinesin-3 motor protein KIF1B has alternatively spliced isoforms distinguished by the presence or absence of insertion sequences in the conserved amino-terminal region of the protein. The insertions are located in the loop region containing the lysine-rich cluster, also known as the K-loop, and in the hinge region adjacent to the motor domain. To clarify the functions of these alternative splice variants of KIF1B, we examined the biochemical properties of recombinant KIF1B with and without insertion sequences. In a microtubule-dependent ATPase assay, KIF1B variants that contained both insertions had higher activity and affinity for microtubules than KIF1B variants that contained no insertions. Mutational analysis of the K-loop insertion revealed that variants with a longer insertion sequence at this site had higher activity. However, the velocity of movement in motility assays was similar between KIF1B with and without insertion sequences. Our results indicate that splicing isoforms of KIF1B that vary in their insertion sequences have different motor activities.

Saccharomyces cerevisiae Na+/H+ antiporter Nha1p associates with lipid rafts and requires sphingolipid for stable localization to the plasma membrane.

The plasma membrane-type Na+/H+ antiporter Nha1p from budding yeast plays an important role in intracellular Na+ and pH homeostasis by mediating the exchange of Na+ for H+ across the plasma membrane. However, the mechanism of intracellular targeting of Nha1p to the plasma membrane remains unknown. Here, we found that Nha1p exists predominantly in detergent-resistant membrane fractions (DRMs) following density gradient centrifugation. When ergosterol was extracted from membranes, Nha1p was transferred to a detergent-soluble fraction, suggesting that Nha1p associates with ergosterol-containing DRMs, also known as lipid rafts. Density gradient centrifugation of cell extracts of yeast mutants that were defective in different stages of the secretory pathway revealed that, unlike previously identified raft proteins, the association of Nha1p with DRMs occurs mainly at the plasma membrane. In lcb1-100 cells, which are temperature-sensitive for sphingolipid synthesis, newly synthesized Nha1p failed to localize to the plasma membrane at the non-permissive temperature. Rather, Nha1p was distributed in an intracellular punctate pattern. The addition of phytosphingosine or the inhibition of endocytosis in lcb1-100 cells restored the targeting of Nha1p to the plasma membrane. The results of the current study suggest that sphingolipids are required for the stable localization of Nha1p to the plasma membrane.

Design, synthesis, and biological evaluation of fluorinated analogues of salicylihalamide.

Salicylihalamide A (SA), a benzolactone enamide compound, possesses potent cytotoxicity against human tumor cell lines. SA is a selective inhibitor of mammalian vacuolar type H(+)-ATPase (V-ATPase), and is distinct from previously known V-ATPase inhibitors such as bafilomycins and concanamycins that do not discriminate between mammalian and nonmammalian V-ATPases. Because of its potent antitumor activity and structural simplicity, SA is a promising candidate for an anticancer drug. Although a number of structure-activity relation studies using synthetic analogues have been reported, no fluorinated derivative of SA has been evaluated even though selective addition of a fluorine atom into a therapeutic small molecule candidate often enhances pharmacokinetic and physicochemical properties. We designed and synthesized fluorinated analogues of SA and evaluated their V-ATPase inhibitory activities. Compared to the natural product, the synthetic analogues were potent V-ATPase inhibitors, suggesting that these analogues are potential drug candidates and potential molecular probes for mode-of-action studies using fluorine-based analytical methods such as (19)F-NMR spectroscopy.

Cell surface levels of organellar Na+/H+ exchanger isoform 6 are regulated by interaction with RACK1.

In mammalian cells, four Na(+)/H(+) exchangers (NHE6 - NHE9) are localized to intracellular compartments. NHE6 and NHE9 are predominantly localized to sorting and recycling endosomes, NHE7 to the trans-Golgi network, and NHE8 to the mid-trans-Golgi stacks. The unique localization of NHEs may contribute to establishing organelle-specific pH values and ion homeostasis in cells. Mechanisms underlying the regulation and targeting of organellar NHEs are largely unknown. We identified an interaction between NHE9 and RACK1 (receptor for activated C kinase 1), a cytoplasmic scaffold protein, by yeast two-hybrid screening using the NHE9 C terminus as bait. The NHE9 C terminus is exposed to the cytoplasm, verifying that the interaction is topologically possible. The binding region was further delineated to the central region of the NHE9 C terminus. RACK1 also bound NHE6 and NHE7, but not NHE8, in vitro. Endogenous association between NHE6 and RACK1 was confirmed by co-immunoprecipitation and co-localization in HeLa cells. The luminal pH of the recycling endosome was elevated in RACK1 knockdown cells, accompanied by a decrease in the amount of NHE6 on the cell surface, although the total level of NHE6 was not significantly altered. These results indicate that RACK1 plays a role in regulating the distribution of NHE6 between endosomes and the plasma membrane and contributes to maintaining luminal pH of the endocytic recycling compartments.

Functional assembly of the Na+/H+ antiporter of Helicobacter pylori from partial fragments in vivo.

Functional assembly of the Helicobacter pylori Na+/H+ antiporter (HPNhaA) from partial fragments was studied. Expression plasmids encoding a series of complementary N- and C-terminal fragment pairs containing the transmembrane domains (TMs) were constructed by inserting a stop or a start codon into each of the loop regions of NhaA. HPNhaA fragments alone or complementary fragment pairs were expressed in DeltanhaA Escherichia coli, and fragment integration into the membrane and antiporter activity were measured. TM1-10, TM1-11, TM2-12, TM6-12, and TM10-12 were found in the membrane fraction, while the other fragments were not. While no single fragment displayed antiporter activity, simultaneous expression of fragments in certain pairs, such as TM1-2 + TM3-12, TM1-8 + TM9-12, or TM1-11 + TM12, reconstituted antiporter activity. With the exception of TM12, all of the fragments in the pairs were detected in the membrane. No single fragments expressed alone for these pairs were found in the membrane, except for TM1-11, suggesting that the interaction between the fragments in these pairs stabilized the fragments and enabled reconstitution of HPNhaA. We also found that the simultaneous expression of three complementary fragments (TM1-2 + TM3-8 + TM9-12) reconstituted HPNhaA activity. Other pairs that were found in the membrane (TM1-5 + TM6-12, TM1-10 + TM11-12, and TM1 + TM2-12) did not reconstitute antiporter activity, suggesting that they may not have the proper conformation. These results revealed that the ability to reconstitute antiporter activity depends on the split position in the loop regions and the interaction between complementary fragment pairs. We propose that formation of the active HPNhaA molecule is initiated by the interaction of short-lived intermediates and maintained by the increased stability of the intermediates within the resulting complex.