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Na+/H+exchanger 5 (NHE5) is enriched in neurons and cycles between recycling endosomes and plasma membranes and transports protons to the endosomal lumen as well as to the extracellular space. Although NHE5 expression is undetectable in normal astrocytes, C6 glioma cells express NHE5 at an elevated level. Using C6 cells as a model, here we demonstrate that NHE5 has an important role in tumor growth and tumor cell proliferation and invasion. Glioma xenografts originating fromNHE5-knockdown cells exhibited significantly slower growth than those fromNHE1-knockdown cells and control cells. Histological characterization of the migration front ofNHE5-knockdown tumors revealed a less invasive and less proliferative appearance thanNHE1-knockdown and control tumors.NHE5-knockdown but notNHE1-knockdown led to downregulation of fetal bovine serum (FBS)-induced MET and EGFR signaling. Moreover, depletion of NHE5 but not NHE1 reduced the ability of cells to spread on collagen. We found that NHE5 depletion greatly abrogated endocytic recycling and the protein stability of β1-integrin, which in part accounted for the defective cell adhesion, spreading, and invasion ofNHE5-knockdown cells.

The role of endosomal pH in neurite formation, one of the principal processes of neuronal differentiation, is unknown. This study shows that the neuron-enriched Na+/H+ exchanger NHE5 potently acidifies recycling endosomes and regulates TrkA trafficking, NGF-TrkA signaling, and neurite outgrowth., To facilitate polarized vesicular trafficking and signal transduction, neuronal endosomes have evolved sophisticated mechanisms for pH homeostasis. NHE5 is a member of the Na+/H+ exchanger family and is abundantly expressed in neurons and associates with recycling endosomes. Here we show that NHE5 potently acidifies recycling endosomes in PC12 cells. NHE5 depletion by plasmid-based short hairpin RNA significantly reduces cell surface abundance of TrkA, an effect similar to that observed after treatment with the V-ATPase inhibitor bafilomycin. A series of cell-surface biotinylation experiments suggests that anterograde trafficking of TrkA from recycling endosomes to plasma membrane is the likeliest target affected by NHE5 depletion. NHE5 knockdown reduces phosphorylation of Akt and Erk1/2 and impairs neurite outgrowth in response to nerve growth factor (NGF) treatment. Of interest, although both phosphoinositide 3-kinase–Akt and Erk signaling are activated by NGF-TrkA, NGF-induced Akt-phosphorylation appears to be more sensitively affected by perturbed endosomal pH. Furthermore, NHE5 depletion in rat cortical neurons in primary culture also inhibits neurite formation. These results collectively suggest that endosomal pH modulates trafficking of Trk-family receptor tyrosine kinases, neurotrophin signaling, and possibly neuronal differentiation.

pH homeostasis in neurons plays crucial roles in normal synaptic functions. It is found that the Na+/H+ exchanger NHE5 is targeted to the synapse on neuronal activation, regulates the synaptic pH, and controls the morphology of dendritic spines., Subtle changes in cellular and extracellular pH within the physiological range have profound impacts on synaptic activities. However, the molecular mechanisms underlying local pH regulation at synapses and their influence on synaptic structures have not been elucidated. Dendritic spines undergo dynamic structural changes in response to neuronal activation, which contributes to induction and long-term maintenance of synaptic plasticity. Although previous studies have indicated the importance of cytoskeletal rearrangement, vesicular trafficking, cell signaling, and adhesion in this process, much less is known about the involvement of ion transporters. In this study we demonstrate that N-methyl-d-aspartate (NMDA) receptor activation causes recruitment of the brain-enriched Na+/H+ exchanger NHE5 from endosomes to the plasma membrane. Concomitantly, real-time imaging of green fluorescent protein–tagged NHE5 revealed that NMDA receptor activation triggers redistribution of NHE5 to the spine head. We further show that neuronal activation causes alkalinization of dendritic spines following the initial acidification, and suppression of NHE5 significantly retards the activity-induced alkalinization. Perturbation of NHE5 function induces spontaneous spine growth, which is reversed by inhibition of NMDA receptors. In contrast, overexpression of NHE5 inhibits spine growth in response to neuronal activity. We propose that NHE5 constrains activity-dependent dendritic spine growth via a novel, pH-based negative-feedback mechanism.

Ni/TiO 2 with bimodal pore structure was prepared from nickel nitrate and titanium alkoxide by a sol–gel process under acidic conditions in the presence of formamide. The Ni/TiO 2 has continuous macropores which are formed by fixing the transitional structure of phase separation. Although the addition of nickel nitrate in the sol–gel reaction of titanium alkoxide steeply decreases the phase separation tendency, the macropore size can be controlled by changing the composition of preparation. The Ni/TiO 2 prepared by sol–gel has higher dispersion of Ni compared with that prepared by impregnation, and shows catalytic activity for the selective hydrogenation of crotonaldehyde: selectivity in the hydrogenation of C C bond is much higher than that of C O bond over sol–gel-derived Ni/TiO 2 .

The ubiquitously expressed Na(+)/H(+) exchanger NHE1 plays an important role in regulating polarized membrane protrusion and directional motility in non-neuronal cells. Using NGF-differentiated PC12 cells and murine neocortical neurons in vitro, we now show that NHE1 plays a role in regulating early neurite morphogenesis. NHE1 was expressed in growth cones in which it gave rise to an elevated intracellular pH in actively extending neurites. The NHE1 inhibitor cariporide reversibly reduced growth cone filopodia number and the formation and elongation of neurites, especially branches, whereas the transient overexpression of full-length NHE1, but not NHE1 mutants deficient in either ion translocation activity or actin cytoskeletal anchoring, elicited opposite effects. In addition, compared with neocortical neurons obtained from wild-type littermates, neurons isolated from NHE1-null mice exhibited reductions in early neurite outgrowth, an effect that was rescued by overexpression of full-length NHE1 but not NHE1 mutants. Finally, the growth-promoting effects of netrin-1, but not BDNF or IGF-1, were markedly reduced by cariporide in wild-type neocortical neurons and were not observed in NHE1-null neurons. Although netrin-1 failed to increase growth cone intracellular pH or Na(+)/H(+) exchange activity, netrin-1-induced increases in early neurite outgrowth were restored in NHE1-null neurons transfected with full-length NHE1 but not an ion translocation-deficient mutant. Collectively, the results indicate that NHE1 participates in the regulation of early neurite morphogenesis and identify a novel role for NHE1 in the promotion of early neurite outgrowth by netrin-1.

Randomly oriented polycrystalline silicon (poly-Si) thin films with a typical grain size of 20×2 µm2 grown by continuous-wave laser lateral crystallization (CLC) were obtained. It was found that CLC poly-Si thin films have a large tensile strain corresponding to 0.6% of single-crystalline Si lattice in the in-plane direction. These results suggest that there is a possibility that not only grain size but also the large tensile strain in in-plane direction can affect thin film transistor (TFT) performance.

NHE7 was identified as the first mammalian organelle-membrane type (Na+, K+)/H+ exchanger that may contribute to the ion homeostasis in the trans-Golgi network (TGN) and endosomes. Here we show that caveolins directly bind to the C-terminal extension of NHE7 by an unconventional binding-module. NHE7 is partly associated with caveolae/lipid raft fractions, and heterologous expression of caveolin dominant-negative mutants as well as cholesterol depriving drugs diminished such associations. In contrast to the wild type NHE7, a deletion mutant lacking the C-terminal extension was predominantly detected in non-caveolae/lipid rafts. We further show that a small fraction of NHE7 is targeted to the cell surface and subsequently internalized. Endocytosis of NHE7 was efficiently inhibited by pharmacological maneuvers that block clathrin-dependent endocytosis, whereas dominant-negative caveolin mutants or methyl β-cyclodextrin did not affect NHE7-internalization. Thus, NHE7 associates with both caveolae/lipid rafts and non-caveolae/lipid raft, and the two pools likely exhibit separate dynamics.

Na+/H+ exchanger isoform 5 (NHE5) is a brain-enriched NHE that may play important roles in ion homeostasis and cell-volume regulation. However, the regulation mechanism of NHE5 has not been fully elucidated. Here, we show that Receptor for Activated C-kinase 1 (RACK1) directly binds to NHE5 and positively regulates the transporter function. NHE5 co-localized with RACK1 as well as β1 integrin, paxillin and vinculin, suggesting that NHE5 associates with focal adhesions. By using RACK1 dominant-negative mutants and siRNA, we further show that RACK1 regulates NHE5 both directly and through an integrin-dependent pathway. The NHE5–RACK1 interaction, but not the RACK1–β1 integrin interaction, was reinforced when cells were spread on an integrin–substrate fibronectin. We propose that RACK1 activates NHE5 both by integrin-dependent and independent pathways, which may coordinate cellular ion homeostasis during cell–matrix adhesion.

The neuron-enriched Na+/H+ exchanger NHE5 is expressed in C6 glioma cells, acidifies recycling endosomes, and modulates cell surface abundance of receptor tyrosine kinases MET and EGFR. NHE5 depletion impairs MET recycling and facilitates degradation, thereby impairing cell migration and polarity., Increased recycling and elevated cell surface expression of receptors serve as a mechanism for persistent receptor-mediated signaling. We show that the neuron-enriched Na+/H+ exchanger NHE5 is abundantly expressed in C6 glioma cells and plays an important part in regulating cell surface expression of the receptor tyrosine kinases MET and EGF receptor. NHE5 is associated with transferrin receptor (TfR)- and Rab11-positive recycling endosomal membranes, and NHE5 knockdown by short hairpin RNA significantly elevates pH of TfR-positive recycling endosomes. We present evidence that NHE5 facilitates MET recycling to the plasma membrane, protects MET from degradation, and modulates HGF-induced phosphatidylinositol-3-kinase and mitogen-activated protein kinase signaling. Moreover, NHE5 depletion abrogates Rac1 and Cdc42 signaling and actin cytoskeletal remodeling. We further show that NHE5 knockdown impairs directed cell migration and causes loss of cell polarity. Our study highlights a possible role of recycling endosomal pH in regulating receptor-mediated signaling through vesicular trafficking.

Secretory Carrier Membrane Proteins (SCAMPs) are a group of tetraspanning integral membrane proteins evolutionarily conserved from insects to mammals and plants. Mammalian genomes contain five SCAMP genes SCAMP1-SCAMP5 that regulate membrane dynamics, most prominently membrane-depolarization and Ca(2+)-induced regulated secretion, a key mechanism for neuronal and neuroendocrine signaling. However, the biological role of SCAMPs has remained poorly understood primarily owing to the lack of appropriate model organisms and behavior assays. Here we generate Drosophila Scamp null mutants and show that they exhibit reduced lifespan and behavioral abnormalities including impaired climbing, deficiency in odor associated long-term memory, and a susceptibility to heat-induced seizures. Neuron-specific restoration of Drosophila Scamp rescues all Scamp null behavioral phenotypes, indicating that the phenotypes are due to loss of neuronal Scamp. Remarkably, neuronal expression of human SCAMP genes rescues selected behavioral phenotypes of the mutants, suggesting the conserved function of SCAMPs across species. The newly developed Drosophila mutants present the first evidence that genetic depletion of SCAMP at the organismal level leads to varied behavioral abnormalities, and the obtained results indicate the importance of membrane dynamics in neuronal functions in vivo.

The neuronal Na + /H + exchanger NHE5 isoform not only resides in the plasma membrane but also accumulates in recycling vesicles by means of clathrin-mediated endocytosis. To further investigate the underlying molecular mechanisms, a human brain cDNA library was screened for proteins that interact with the cytoplasmic C-terminal region of NHE5 by using yeast two-hybrid methodology. One candidate cDNA identified by this procedure encoded β-arrestin2, a specialized adaptor/scaffolding protein required for internalization and signaling of members of the G protein-coupled receptor superfamily. Direct interaction between the two proteins was demonstrated in vitro by GST fusion protein pull-down assays. Sequences within the N - terminal receptor activation-recognition domain and the C-terminal secondary receptor-binding domain of β-arrestin2 conferred strong binding to the C terminus of NHE5. Full-length NHE5 and β-arrestin2 also associated in intact cells, as revealed by their coimmunoprecipitation from extracts of transfected CHO cells. Moreover, ectopic expression of both proteins caused a redistribution of β-arrestin2 from the cytoplasm to vesicles containing NHE5, and significantly decreased the abundance of the transporter at the cell surface. Comparable results were also obtained for the β-arrestin1 isoform. These data reveal a broader role for arrestins in the trafficking of integral plasma membrane proteins than previously recognized.

Mammalian Na+/H+ exchangers (NHEs) are a family of integral membrane proteins that play central roles in sodium, acid-base, and cell volume homeostasis. The recently cloned NHE5 isoform is expressed predominantly in brain, but its functional and cellular properties are poorly understood. To facilitate its characterization, an epitope-tagged construct of NHE5 was ectopically expressed in nonneuronal and neuronal cells. In NHE-deficient Chinese hamster ovary AP-1 cells, NHE5 localized at the plasmalemma, but a significant fraction accumulated intracellularly in vesicles that concentrated in a juxtanuclear region. Similarly, in nerve growth factor-differentiated neuroendocrine PC12 cells and primary hippocampal neurons, immunolabeling of NHE5 was detected in endomembrane vesicles in the perinuclear region of the cell body but also along the processes. More detailed characterization in AP-1 cells using organelle-specific markers showed that NHE5 co-localized with internalized transferrin, a marker of recycling endosomes. Transient transfection of a dominant negative mutant of dynamin-1, which inhibits clathrin-mediated endocytosis, blocked uptake of transferrin as well as internalization of NHE5. Likewise, wortmannin inhibition of phosphatidylinositol 3'-kinase, a lipid kinase implicated in endosomal traffic, induced coalescence of vesicles containing NHE5 and caused a pronounced inhibition of plasmalemmal Na+/H+ exchange. By contrast, disruption of the F-actin cytoskeleton with cytochalasin D increased cell surface NHE5 activity and abundance. These observations demonstrate that NHE5 is localized to the recycling endosomal pathway and is dynamically regulated by phosphatidylinositol 3'-kinase and by the state of F-actin assembly.

Neuronal precursor cells extend multiple neurites during development, one of which extends to form an axon whereas others develop into dendrites. Chemical stimulation of N-methyl D-aspartate (NMDA) receptor in fully-differentiated neurons induces projection of dendritic spines, small spikes protruding from dendrites, thereby establishing another layer of polarity within the dendrite. Neuron-enriched Na(+)/H(+) exchanger NHE5 contributes to both neurite growth and dendritic spine formation. In resting neurons and neuro-endocrine cells, neuron-enriched NHE5 is predominantly associated with recycling endosomes where it colocalizes with nerve growth factor (NGF) receptor TrkA. NHE5 potently acidifies the lumen of TrkA-positive recycling endosomes and regulates cell-surface targeting of TrkA, whereas chemical stimulation of NMDA receptors rapidly recruits NHE5 to dendritic spines, alkalinizes dendrites and down-regulates the dendritic spine formation. Possible roles of NHE5 in neuronal signaling via proton movement in subcellular compartments are discussed.

The luminal pH of organelles along the secretory and endocytic pathways of mammalian cells is acidic and tightly regulated, with the [H+] varying up to 100-fold between compartments. Steady-state organellar pH is thought to reflect a balance between the rates of H+ pumping by the vacuolar-type H+-ATPase and H+ efflux through ill-defined pathways. Here, we describe the cloning of a novel gene (NHE7) in humans that is homologous to Na+/H+ exchangers, is ubiquitously expressed, and localizes predominantly to the trans-Golgi network. Significantly, NHE7 mediates the influx of Na+ or K+ in exchange for H+. The activity of NHE7 was also found to be relatively insensitive to inhibition by amiloride but could be antagonized by the analogue benzamil and the unrelated compound quinine. Thus, NHE7 displays unique functional and pharmacological properties and may play an important role in maintaining cation homeostasis of this important organelle.

The electroneutral exchange of protons for Na+ and K+ across the mitochondrial inner membrane contributes to organellar volume and Ca2+ homeostasis. The molecular nature of these transporters remains unknown. In this report, we characterize a novel gene (YDR456w; renamed NHA2) in Saccharomyces cerevisiae whose deduced protein sequence is homologous to members of the mammalian Na+/H+ exchanger gene family. Fluorescence microscopy showed that a Nha2-green fluorescent protein chimera colocalizes with 4',6-diamidino-2-phenylindole staining of mitochondrial DNA. To assess the function of Nha2, we deleted the NHA2 gene by homologous disruption and found that benzamil-inhibitable, acid-activated 22Na+ uptake into mitochondria was abolished in the mutant strain. It also showed retarded growth on nonfermentable carbon sources and severely reduced survival during the stationary phase of the cell cycle compared with the parental strain, consistent with a defect in aerobic metabolism. Sequence comparisons revealed that Nha2 has highest identity to a putative Na+/H+ exchanger homologue (KIAA0267; renamed NHE6) in humans. Northern blot analysis demonstrated that NHE6 is ubiquitously expressed but is most abundant in mitochondrion-rich tissues such as brain, skeletal muscle, and heart. Fluorescence microscopy showed that a NHE6-green fluorescent protein chimera also accumulates in mitochondria of transfected HeLa cells. These data indicate that NHA2 and NHE6 encode homologous Na+/H+ exchangers and suggest they may be important for mitochondrial function in lower and higher eukaryotes, respectively.

Some neurotrophic factors stimulate process outgrowth in peripheral and/or central nerve fibres. There is no published report that has focused on the expression of neurotrophic factors in human pancreatic cancer except basic fibroblast growth factor. This study was therefore designed to examine the mRNA and protein levels of neurotrophin-3 (NT-3), which is one of the representative neurotrophic factors. The mRNA level for NT-3 was first investigated in eight pancreatic cancers and two samples of normal pancreas, using reverse transcription-polymerase chain reaction (RT-PCR). NT-3 protein expression was then studied in 47 human pancreatic cancers, using a monoclonal antibody against human NT-3 protein. There was a notable difference in the level of NT-3 mRNA between normal and pancreatic carcinoma tissues, with no evident difference in the expression of the beta-actin gene. Immunohistochemically, 34 of 47 pancreatic cancers (72 per cent) were mildly to markedly immunoreactive for NT-3 in the cytoplasma. Immunoreactivity was usually more pronounced at the infiltrative margins of the tumours. These observations suggest that pancreatic carcinoma overexpresses NT-3.

金沢大学医薬保健研究域医学系, We examined the expression of basic fibroblast growth factor (FGF) and FGF receptor by immunohistochemistry in 32 human pancreatic ductal adenocarcinomas. Mild to marked basic FGF immunoreactivity was noted in 19 (59.4%) of the 32 tumours examined, and 30 (93.3%) of the tumours exhibited a cytoplasmic staining pattern against FGF receptor. The tumours were divided into two groups according to the proportion of positively stained tumour cells: A low expression group (positive cells ≪ 25%) and a high expression group (positive cells > or = 25%). No statistically significant difference in tumour size, differentiation, metastases or stage was found between the low and high basic FGF expression groups. However, a significant correlation was found between FGF receptor expression level and the presence of retroperitoneal invasion, lymph node metastasis, and tumour stage. In addition, low FGF receptor expression was significantly associated with a longer post-operative survival as compared with high FGF receptor expression, whereas there was no significant difference in post-operative survival between the low and high basic FGF expression groups. Increased expression of FGF receptor is correlated with the extent of malignancy and post-operative survival in human pancreatic ductal adenocarcinomas. Thus, overexpression of FGF receptor may prove to be a more useful prognostic marker than basic FGF expression level in pancreatic cancer patients. © 1995 Stockton Press. All rights reserved., Embargo Period 12 months

Vacuolar H(+)-ATPase (V-ATPase), an enzyme composed of multisubunits, is located in the membrane of intracellular organelles (e.g., lysosomes, and endosomes) and maintains the intraorganellar acidic pH by pumping protons across the membrane. Although there is growing evidence for some important role of V-ATPase in cell proliferation and differentiation, the functional significance of V-ATPase in vivo during mammalian development remains obscure. In the present study we investigated the expression and localization of mRNA for the 16 KD subunit of V-ATPase, an essential sector for enzymatic activity, in prenatal rat by Northern blot analysis and in situ hybridization with a specific oligonucleotide probe. With Northern blot analysis, consistent expression of the mRNA was observed in the embryos throughout the period examined (E14-E20). On in situ hybridization, mRNA signal was distributed with various intensities in both the epithelial and mesenchymal tissues at embryonic day 14 (E14). In E17 and E20 embryos, localization of strong signal became more restricted to distinct mesenchymal cells such as fibroblasts adjacent to the epithelia of skin, lung, and intestine, the cells of perichondrium, and myoblasts in the process of fusion. These results suggest that V-ATPase performs specific functions during the later stages of embryogenesis, especially at sites of mesenchymal differentiation and epithelium-mesenchyme interaction.

Mammalian target of rapamycin complex 1 (mTORC1) signaling is frequently dysregulated in cancer. Inhibition of mTORC1 is thus regarded as a promising strategy in the treatment of tumors with elevated mTORC1 activity. We have recently identified niclosamide (a Food and Drug Administration-approved antihelminthic drug) as an inhibitor of mTORC1 signaling. In the present study, we explored possible mechanisms by which niclosamide may inhibit mTORC1 signaling. We tested whether niclosamide interferes with signaling cascades upstream of mTORC1, the catalytic activity of mTOR, or mTORC1 assembly. We found that niclosamide does not impair PI3K/Akt signaling, nor does it inhibit mTORC1 kinase activity. We also found that niclosamide does not interfere with mTORC1 assembly. Previous studies in helminths suggest that niclosamide disrupts pH homeostasis of the parasite. This prompted us to investigate whether niclosamide affects the pH balance of cancer cells. Experiments in both breast cancer cells and cell-free systems demonstrated that niclosamide possesses protonophoric activity in cells and in vitro. In cells, niclosamide dissipated protons (down their concentration gradient) from lysosomes to the cytosol, effectively lowering cytoplasmic pH. Notably, analysis of five niclosamide analogs revealed that the structural features of niclosamide required for protonophoric activity are also essential for mTORC1 inhibition. Furthermore, lowering cytoplasmic pH by means other than niclosamide treatment (e.g. incubation with propionic acid or bicarbonate withdrawal) recapitulated the inhibitory effects of niclosamide on mTORC1 signaling, lending support to a possible role for cytoplasmic pH in the control of mTORC1. Our data illustrate a potential mechanism for chemical inhibition of mTORC1 signaling involving modulation of cytoplasmic pH.

A major role of DNA-methyltransferase (MTase) is thought to be maintaining the DNA methylation profile through DNA replication. However, previous surveys of mRNA distribution in different tissues by Northern-blot analysis have shown unexpectedly high levels of expression of DNA-MTase mRNA in adult mouse brain, which consists mostly of slowly proliferating glial and nonproliferating neuronal cells. In order to identify cells expressing the gene in the brain, we performed an in situ hybridization analysis of mature brain as well as whole embryos of different ages. In addition to various embryonic tissues with active cell proliferation such as the ventricular neurogenic layer, hair follicle epithelia, thymus and epithelia of the base of intestinal villi, almost all mature neurons in brain of adult and even aged mice expressed DNA-MTase mRNA at substantial levels. No significant expression of the gene was detected in the white matter. These findings suggest some neuron-specific biological function of DNA methylation, unrelated to DNA replication.

Na+/H+ exchangers (NHEs) are a group of secondary active antiporters that regulate cellular pH, cell volume and ion homeostasis. In humans, nine isoforms (NHE1-NHE9) were identified and characterized as functional NHEs. While a growing body of evidence indicates that NHE1 generates an acidic tumor environment and thereby contributes to tumor invasion, little is known about the role of other NHE isoforms in tumor progression. NHE7 is a unique member of the NHE gene family that dynamically shuttles between the trans-Golgi network, endosomes and the plasma membrane, and regulates the luminal pH of these organelles. Here we show that NHE7-overexpression in breast cancer MDA-MB-231 cells enhances cell overlay, cell-cell adhesion, invasion, anchorage-independent tumor growth and tumor formation in vivo. In contrast, NHE1-overexpression enhances tumor invasion, but it has little effect on cell adhesion or anchorage-independent tumor growth. Pathological examinations of the tumor samples derived from NHE7-overexpressing cells showed a similar appearance to aggressive tumors. Together, these results suggest that NHE7 enhances tumor progression. This is the first report to show the involvement of an organellar NHE in oncogenic processes.

Pancreatic trypsinogen expression in 149 surgically resected extrapancreatic gastrointestinal neoplasms was evaluated immunohistochemically. Immunohistochemistry was performed using a monoclonal antibody against human pancreatic trypsinogen. Pancreatic trypsinogen expression was detected in 28 of 55 gastric carcinomas (50.9%), 22 of 44 colorectal cancers (50%), 12 of 20 gallbladder cancers (60%), nine of 10 extrahepatic bile duct cancers (90%), and none of 20 hepatocellular carcinomas. The intensity of immunoreactivity in the tumor area varied from specimen to specimen, and from area to area within the same specimen. In most cases, however, immunoreactivity was more pronounced at the infiltrative margin of the tumor. Additionally, the highly differentiated carcinoma cells tended to display a focal, fine granular immunoreactive pattern, usually present in the supranuclear cytoplasm, while the poorly differentiated carcinoma cells displayed a fine granular pattern, usually present over the entire cytoplasm. These findings suggest that some extrapancreatic gastrointestinal neoplasms express pancreatic trypsinogen immunoreactive peptides, raising the possibility that secreted pancreatic trypsinogen plays a role in carcinoma invasion and metastasis, as has been shown for other classes of proteases.

Background Acidification of the cytoplasm and the extracellular environment is associated with many physiological and pathological conditions, such as intense exercise, hypoxia and tumourigenesis. Acidification affects important cellular functions including protein synthesis, growth, and proliferation. Many of these vital functions are controlled by mTORC1, a master regulator protein kinase that is activated by various growth-stimulating signals and inactivated by starvation conditions. Whether mTORC1 can also respond to changes in extracellular or cytoplasmic pH and play a role in limiting anabolic processes in acidic conditions is not known. Methodology/Findings We examined the effects of acidifying the extracellular medium from pH 7.4 to 6.4 on human breast carcinoma MCF-7 cells and immortalized mouse embryo fibroblasts. Decreasing the extracellular pH caused intracellular acidification and rapid, graded and reversible inhibition of mTORC1, assessed by measuring the phosphorylation of the mTORC1 substrate S6K. Fibroblasts deleted of the tuberous sclerosis complex TSC2 gene, a major negative regulator of mTORC1, were unable to inhibit mTORC1 in acidic extracellular conditions, showing that the TSC1–TSC2 complex is required for this response. Examination of the major upstream pathways converging on the TSC1–TSC2 complex showed that Akt signaling was unaffected by pH but that the Raf/MEK/ERK pathway was inhibited. Inhibition of MEK with drugs caused only modest mTORC1 inhibition, implying that other unidentified pathways also play major roles. Conclusions This study reveals a novel role for the TSC1/TSC2 complex and mTORC1 in sensing variations in ambient pH. As a common feature of low tissue perfusion, low glucose availability and high energy expenditure, acidic pH may serve as a signal for mTORC1 to downregulate energy-consuming anabolic processes such as protein synthesis as an adaptive response to metabolically stressful conditions.

NHE5 is a brain-enriched Na+/H+ exchanger that dynamically shuttles between the plasma membrane and recycling endosomes, serving as a mechanism that acutely controls the local pH environment. In the current study we show that secretory carrier membrane proteins (SCAMPs), a group of tetraspanning integral membrane proteins that reside in multiple secretory and endocytic organelles, bind to NHE5 and co-localize predominantly in the recycling endosomes. In vitro protein-protein interaction assays revealed that NHE5 directly binds to the N- and C-terminal cytosolic extensions of SCAMP2. Heterologous expression of SCAMP2 but not SCAMP5 increased cell-surface abundance as well as transporter activity of NHE5 across the plasma membrane. Expression of a deletion mutant lacking the SCAMP2-specific N-terminal cytosolic domain, and a mini-gene encoding the N-terminal extension, reduced the transporter activity. Although both Arf6 and Rab11 positively regulate NHE5 cell-surface targeting and NHE5 activity across the plasma membrane, SCAMP2-mediated surface targeting of NHE5 was reversed by dominant-negative Arf6 but not by dominant-negative Rab11. Together, these results suggest that SCAMP2 regulates NHE5 transit through recycling endosomes and promotes its surface targeting in an Arf6-dependent manner.

Organellar and cytosolic pH homeostasis is central to most cellular processes, including vesicular trafficking, post-translational modification/processing of proteins, and receptor-ligand interactions. SLC9A7 (NHE7) was identified as a unique (Na+, K+)/H+ exchanger that dynamically cycles between the trans-Golgi network (TGN), endosomes and the plasma membrane. Here we have used mass spectrometry to explore the affinity-captured interactome of NHE7, leading to the identification of cytoskeletal proteins, cell adhesion molecules, membrane transporters, and signaling molecules. Among these binding proteins, calcium-calmodulin, but not apo-calmodulin, binds to NHE7 and regulates the organellar transporter activity. Vimentin was co-immunoprecipitated with endogenous NHE7 protein in human breast cancer MDA-MB-231 cells. A sizable population of NHE7 relocalized to focal complexes in migrating cells and showed colocalization with vimentin and actin in focal complexes. Among the NHE7-binding proteins identified, CD44, a cell surface glycoprotein receptor for hyaluronate and other ligands, showed regulated interaction with NHE7. Pretreatment of the cells with phorbol ester facilitated the NHE7-CD44 interaction and the lipid raft association of CD44. When lipid rafts were chemically disrupted, the NHE7-CD44 interaction was markedly reduced. These results suggest potential dual roles of NHE7 in intracellular compartments and subdomains of cell-surface membranes.

The mammalian (Na+,K+)/H+ exchanger NHE7 resides chiefly in the trans-Golgi network (TGN) and post-Golgi vesicles where it is thought to contribute to organellar pH homeostasis. However, the mechanisms that underlie the targeting and regulation of NHE7 are unknown. To gain insight into these processes, yeast two-hybrid methodology was used to screen a human brain cDNA library for proteins that interact with the cytoplasmic C-terminus of NHE7. One binding partner we identified was SCAMP2, a member of the secretory carrier membrane protein (SCAMP) gene family. Direct association of these two proteins was further supported by co-immunolocalization and co-immunoprecipitation analyses using transfected cells, by their co-sedimentation in membrane fractions resolved on sucrose density gradients, and by in vitro protein binding assays. Other members of the SCAMP family, such as SCAMP1 and SCAMP5, also associated with NHE7. The majority of the NHE7-SCAMP complexes accumulated at the TGN, but a minor fraction also resided in recycling vesicles. Biochemical analyses indicated that the C-terminal cytoplasmic tail of NHE7 bound preferentially to a highly conserved cytoplasmic loop between the second and the third transmembrane segments (TM2-TM3 loop) of SCAMP2. A deletion mutant of SCAMP2 lacking this region (SCAMP2/Δ184-208) bound weakly to NHE7, but caused a significant fraction of NHE7 and wild-type SCAMP2 to redistribute to a pool of scattered recycling vesicles without noticeably affecting the location of other resident TGN (syntaxin 6) or Golgi cisternae (GM130) proteins. Conversely, a GFP-tagged TM2-TM3 construct of SCAMP2 interacted with NHE7, but also led to the redistribution of NHE7 to dispersed vesicular structures. We propose a model wherein SCAMPs participate in the shuttling of NHE7 between recycling vesicles and the TGN.

Mammalian Na+/H+ exchangers (NHEs) are a family of integral membrane proteins that play central roles in sodium, acid-base, and cell volume homeostasis. The recently cloned NHE5 isoform is expressed predominantly in brain, but its functional and cellular properties are poorly understood. To facilitate its characterization, an epitope-tagged construct of NHE5 was ectopically expressed in nonneuronal and neuronal cells. In NHE-deficient Chinese hamster ovary AP-1 cells, NHE5 localized at the plasmalemma, but a significant fraction accumulated intracellularly in vesicles that concentrated in a juxtanuclear region. Similarly, in nerve growth factor-differentiated neuroendocrine PC12 cells and primary hippocampal neurons, immunolabeling of NHE5 was detected in endomembrane vesicles in the perinuclear region of the cell body but also along the processes. More detailed characterization in AP-1 cells using organelle-specific markers showed that NHE5 co-localized with internalized transferrin, a marker of recycling endosomes. Transient transfection of a dominant negative mutant of dynamin-1, which inhibits clathrin-mediated endocytosis, blocked uptake of transferrin as well as internalization of NHE5. Likewise, wortmannin inhibition of phosphatidylinositol 3'-kinase, a lipid kinase implicated in endosomal traffic, induced coalescence of vesicles containing NHE5 and caused a pronounced inhibition of plasmalemmal Na+/H+ exchange. By contrast, disruption of the F-actin cytoskeleton with cytochalasin D increased cell surface NHE5 activity and abundance. These observations demonstrate that NHE5 is localized to the recycling endosomal pathway and is dynamically regulated by phosphatidylinositol 3'-kinase and by the state of F-actin assembly.

金沢大学医薬保健研究域医学系, 金沢大学附属病院脳神経外科, The relationship between the grade of astrocytic tumor and the expression of deoxyribonucleic acid methyltransferase (DNA-MTase) gene was examined. The levels of DNA-MTase messenger ribonucleic acid (mRNA) were measured by semiquantitative reverse transcriptase-polymerase chain reaction in surgical specimens from 12 astrocytic tumors (4 astrocytomas, 6 anaplastic astrocytomas, and 2 glioblastomas) and two normal brain tissues, and in four glioma cell lines. Compared to normal brain tissues, the levels of DNA-MTase mRNA were increased by 16- to 55-fold in low grade astrocytomas, and significantly increased by 200- to 4500-fold in high grade astrocytomas (anaplastic astrocytomas and glioblastomas) and more than 4500-fold in glioma cell lines. In situ hybridization with paraffin-embedded surgical specimens of human astrocytic tumors showed DNA-MTase mRNA was abundantly expressed in high grade astrocytomas. The detection of increased DNA-MTase expression in astrocytic tumor indicates involvement in the tumorigenesis and suggests that blocking of this change with specific inhibitors may offer new therapeutic strategies for malignant astrocytic tumors.

The recently cloned Na(+)/H(+) exchanger isoform 5 (NHE5) is expressed predominantly in brain, yet little is known about its functional properties. To facilitate its characterization, a full-length cDNA encoding human NHE5 was stably transfected into NHE-deficient Chinese hamster ovary AP-1 cells. Pharmacological analyses revealed that H(+)(i)-activated (22)Na(+) influx mediated by NHE5 was inhibited by several classes of drugs (amiloride compounds, 3-methylsulfonyl-4-piperidinobenzoyl guanidine methanesulfonate, cimetidine, and harmaline) at half-maximal concentrations that were intermediate to those determined for the high affinity NHE1 and the low affinity NHE3 isoforms, but closer to the latter. Kinetic analyses showed that the extracellular Na(+) dependence of NHE5 activity followed a simple hyperbolic relationship with an apparent affinity constant (K(Na)) of 18.6 +/- 1.6 mM. By contrast to other NHE isoforms, NHE5 also exhibited a first-order dependence on the intracellular H(+) concentration, achieving half-maximal activation at pH 6.43 +/- 0.08. Extracellular monovalent cations, such as H(+) and Li(+), but not K(+), acted as effective competitive inhibitors of (22)Na(+) influx by NHE5. In addition, the transport activity of NHE5 was highly dependent on cellular ATP levels. Overall, these functional features distinguish NHE5 from other family members and closely resemble those of an amiloride-resistant NHE isoform identified in hippocampal neurons.

We examined whether human pancreatic ductal cancer cells express and secrete pancreatic cationic trypsinogen in vitro which can be spontaneously converted into active trypsin at acidic pH (pH 4.5-5. 5), in contrast to anionic trypsinogen. Cationic trypsinogen expression at the mRNA level was observed in differentiated Capan-1 and BxPC-3 cell lines. However, expression was not detected in either poorly-differentiated Panc-1 or undifferentiated MIAPaCa-2 cell line. The gelatinolytic activity of the activated form of trypsinogen in each conditioned medium in the presence of enterokinase (1.0 microg/ml) (a band with a molecular weight of approximately 23 kDa) corresponded well to the level of cationic trypsinogen mRNA. The spontaneous activation of trypsinogen also was observed by gelatin zymography of the acid-loaded conditioned medium (pH 5.5). These findings suggest that trypsinogen produced by human pancreatic ductal cancer has the characteristics of spontaneous activation and gelatinolytic activity in the presence of proton.

Bafilomycin A1, a specific inhibitor of vacuolar type H(+)-ATPase, can inhibit the growth of a variety of cultured cells in a dose-dependent manner, but its mechanism is unclear. The aim of this study was to examine whether bafilomycin A1 inhibits the growth of Capan-1 human pancreatic cancer cells through apoptosis. The effect of bafilomycin A1 on tumour growth in vitro and in vivo was examined using an MTT assay and an in vivo tumour model. The presence or absence of apoptosis was determined by morphology and DNA analysis of tumour cells. The concentration of bafilomycin A1 for 50 per cent inhibition of cell viability during 72 h by the MTT assay was 5 nm. In DNA analysis, a ladder of fragmented DNA was detected in Capan-1 cells treated with bafilomycin A1 at concentrations greater than 10 nm for 24 h. Nude mice bearing a xenografted Capan-1 cell line tumour received 4 weeks of bafilomycin A1 (1.0 mg/kg per day). This treatment significantly inhibited tumour growth compared with controls after 21 days (P < 0.05). Histopathological examination of tumour cells in the treated group demonstrated signs of apoptosis with chromatin condensation and cell shrinkage. These observations suggest that bafilomycin A1 inhibits the growth of Capan-1 human pancreatic cancer cells through apoptosis.

Recent studies have shown that bafilomycin A(1)-sensitive vacuolar type H+-ATPase (V-ATPase) is responsible for the acidification of intracellular compartments in eukaryotic cells. B-cells of pancreatic islets also are known to include acidifying secretory vesicles which are the major cellular site of proinsulin to insulin conversion. This study was designed to examine immunohistochemically the level of V-ATPase protein expression in normal pancreas (five cases) and benign insulinoma (six cases), using mouse monoclonal antibody raised against the 116 kDa subunit of human V-ATPase. Light microscopic immunohistochemistry revealed that moderate to marked V-ATPase expression was observed in normal islet B-cells, while insulinoma cells in each case expressed V-ATPase faintly or not at all. By immunoelectron microscopy, the majority of secretory vesicles in insulinoma cells did not express V-ATPase protein at their endomembranes, although mild to marked V-ATPase expression was noted at the endomembrane of secretory vesicles in normal islet B-cells. Thus, differential expression of V-ATPase protein at the endomembrane of secretory vesicles was observed between normal islet B-cells and insulinoma cells. These findings suggest that the reduced activity of V-ATPase per insulin secretory vesicle in insulinoma cells have a profound effect on the efficiency of proteolytic cleavage of proinsulin.

Recent studies have shown that bafilomycin A1-sensitive vacuolar-type H(+)-ATPase (V-ATPase) plays important roles in cell growth and differentiation. However, there is no published study that has focused on the expression of V-ATPase in human tumour tissues. This study was designed to examine the mRNA and protein levels for the 16 kilodalton (kDa) proteolipid of V-ATPase in human pancreatic carcinoma tissues. We first investigated the mRNA level for V-ATPase in six cases of invasive pancreatic cancers and two normal pancreases, using reverse transcription-polymerase chain reaction technique. Then, we examined immunohistochemically the level of V-ATPase protein in 49 pancreatic cancers and ten benign cystic neoplasms of the pancreas, using antisera raised against the 16 kDa proteolipid. There was a notable difference in the level of V-ATPase mRNA between normal and pancreatic carcinoma tissues, with no evident difference in the expression of the beta-actin gene. Immunohistochemically, 42 out of 46 invasive ductal cancers (92%) displayed a mild to marked immunoreactivity for V-ATPase in the cytoplasm, whereas neither non-invasive ductal cancers nor benign cystic neoplasms expressed detectable immunoreactive proteins. These findings suggest that the overexpression of V-ATPase protein is characteristic of invasive pancreatic tumours. V-ATPase may play some crucial roles in tumour progression. Images Figure 2 Figure 4 Figure 3

Our previous study demonstrated that apolipoprotein A-1 (apo A- 1) immunoreactive peptides were located diffusely in the cytoplasm, not only of human normal hepatocytes, but also of intrahepatic bile ducts and peribiliary glands. It is important to determine whether the presence of these immunoreactive peptides in intrahepatic biliary tree is caused by pinocytosis from the bile, or by intracellular protein synthesis. Thus, we investigated whether apo A-1 is synthesized by cells that line the biliary tree. Normal human liver samples obtained at surgery were used, and the expression and distribution of apo A-1 mRNA in normal human liver tissues were examined, using in situ hybridization histochemistry with a 35S-labeled oligonucleotide probe specific for apo A-I. On the autoradio- gram, many silver grains were found to be distributed uniformly in hepato- cytes. In addition, an appreciable apo A-1 mRNA signal was also ob- served in both the surface epithelial lining of the bile ducts and the epithelial cells of the peribiliary glands. In conclusion, these findings sug- gest that the apo A-1 found in bile is secreted both by hepatocytes and by I intrahepatic bile duct cells and peribiliary glands.

The 16 kD subunit of vacuolar H(+)-ATPase (V-ATPase) is essential for pumping protons into intracellular organella. Northern blot analysis revealed that the 16 kD subunit of V-ATPase is abundantly expressed in rat brain, and that the amount of transcripts is higher in the postnatal 15 days than in the adult age. On in situ hybridization, the mRNA signal for 16 kD subunit of V-ATPase was detected widely over the brain tissues of both adult and postnatal rats, with higher intensity in most of the gray matters located in the cerebellar cortex, hippocampus, thalamic nuclei, pontin nuclei, and olfactory bulb. In contrast, the signal in the cerebral cortex was preferentially enriched in the surface layers in the postnatal 10 days but was distributed diffusely and weakly over the entire layers thereafter. In all regions of brain, the mRNA signal was located predominantly in the neurons rather than in the glial components.

NUMATA, M., HATA, H., TOHDA, H., YASUI, A and OIKAWA, A. DNA Repair Pathways in Mammalian Cells Analyzed by Isolation of ACNU-Sensitive Chinese Hamster Ovary Cells. Tohoku J. Exp. Med., 1992, 168 (2), 123-128 - 1-[(4- amino-2-methyl-5-pyrimidinyl)methyl]-3-(2-chloroethyl)-3-nitrosourea hydrochloride (ACNU) causes chloroethylation of DNA strand followed by cross linking through an ethylene bridge. We recently isolated two ACNU sensitive mutants from mutagenized Chinese hamster ovary cells, and found them to be new drug sensitive recessive mutansts (Hata et al. 1991). The O6-methyl guanine DNA methyl transferase (MT) activities of these cells were undetectable as the parental cell line, indicating that the sensitivity of the mutant cell lines to ACNU was not due to the decreased cellular level of this enzyme. By complementation analysis with the 7 established UV-sensitive CHO cell lines, one of the mutants, UVS1, turned out to complement their UV-sensitivity and, therefore, build a new complementation group among all the CHO cell lines ever reported. The other mutant, CNU1 showed hypersensitivity only to chloroethylating agents (ACNU, CCNU) and exhibited a slightly reduced unscheduled DNA synthesis (UDS) induced by UV. It is, therefore, suggestive that this mutant is defective in a specific step of DNA repair systems, which is important for the processing of DNA damages produced by ACNU. Only cell lines from the complementation group 1 and 4 out of 7 established complementation groups of UV-sensitive CHO mutants were more sensitive to ACNU than UVS1 and CNU1, indicating some steps of excision repair pathways as well as specific repair system play important roles in repairing ACNU-induced DNA damages. - chloroethylating agent; ACNU; cross linking; DNA repair; CHO mutant

In continuous-wave (cw) laser lateral crystallization of amorphous silicon (a-Si) thin films, the effects of cap SiO2 thin films were investigated. The thickness of the cap SiO2 film was 10 nm, which is sufficiently thin to exclude anti reflection effect. The cap SiO2 thin films suppressed the generation of voids during the cw laser crystallization, and the available crystallization condition to form lateral-crystallized polycrystalline silicon (poly-Si) thin films was expanded. By using the cap SiO2 thin films, the surface of the lateral-crystallized poly-Si thin films became smooth, and an average roughness of 1.3 nm was achieved.

Continuous-wave (CW) laser lateral crystallized silicon (Si) thin films were investigated. CW laser with a wavelength λ of 532 nm was applied to crystallization. In the CW laser lateral crystallization (CLC), a gradual temperature slope in Si thin films was formed and one-dimensionally long grains with a typical length of 20 µm were achieved. The most dominant grain growth direction was direction. ∑3, 9, and 11 boundaries were mainly observed between adjacent grains. It was found that crystallization was performed to form stable grain boundaries. It was also clarified that CLC poly-Si thin films have large tensile strain corresponding to 0.6% of single crystalline Si lattice in in-plane direction. This strain was induced by the difference in thermal expansion coefficients between Si thin films and the buffer SiO2 films.

By use ofin situ hybridization with a35S-labeled oligonucleotide probe, the mRNA for neurofilament protein was localized to the trigeminal ganglion cells of rats. The result was visualized with radioautography at both light and electron microscopic levels. For the latter purpose, the cryostat sections were treated forin situ hybridization prior to embedding in epoxy resin. The presence or absence of detergent in the hybridization solution proved to be critical for the electron-microscopicin situ hybridization in terms of the signal/background ratio and ultrastructural integrity.

A polyclonal antibody (anti-PepT1/C) was raised against the rabbit intestinal H+-coupled oligopeptide transporter, PepT1. Anti-PepT1/C detected 70–80-kDa protein in crude membranes obtained from rabbit duodenum, jejunum and ileum. PepT1 was localized in the brush-border of the absorptive epithelial cells by subcellular fractionation of membranes on a sucrose density gradient and by immunohistochemistry using light and electron microscopy. Transport activity for cephalosporins and dipeptide expressed in Xenopus laevis oocytes injected with total mRNA obtained from rabbit small intestine was eliminated completely by prehybridization of the mRNA with antisense oligonucleotide against the 5′-coding region of rabbit PepT1 cDNA.