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PubMed1998

Carbonic anhydrase II plays a major role in osteoclast differentiation and bone resorption by effecting the steady state intracellular pH and Ca2+.

Carbonic anhydrase II (CA II) expression in characteristic for the early stage of osteoclast differentiation. To study how CA II, which is crucial in proton generation in mature osteoclasts, influences the osteoclast differentiation process we performed rat bone marrow cultures. In this model, acetazolamide, a specific CA inhibitor, decreased the 1,25 (OH)2D3-induced formation of multinucleated tartrate-resistant acid phosphatase (TRAP)-positive cells, in a dose-dependent manner. We then performed intracellular pH (pHi) and Ca2+ (Cai2+) measurements for cultured osteoclasts and noticed that addition of acetazolamide caused a rapid, transient increase of both parameters. The increase in pHi was dependent neither on the culture substrate nor on the extracellular pH (pHe) but the increase could be diminished by DIDS or by bicarbonate removal. Membrane-impermeable CA inhibitors (benzolamide and pd5000) did not have this effect. Addition of CA II antisense oligonucleotides into the cultures reduced the pHi increase significantly. CA II inhibition was also found to neutralize the intracellular vesicles at extracellular pH (pHe) of 7.4, but at less extent at pHe 7.0. In mouse calvaria cultures, bone resorption was inhibited dose dependently by acetazolamide at pHe 7.4 while inhibition was smaller at pHe 7.0. We conclude that CA II is essential not only in bone resorption but also in osteoclast differentiation. In both processes, however, the crucial role of CA II is at least partially due to the effect on the osteoclast pHi regulation.

Acetazolamide↗

PubMed1989

The transverse location of the retinal chromophore in the purple membrane by diffusion-enhanced energy transfer.

We have used fluorescence energy transfer in the rapid-diffusion limit (RDL) to estimate the trans-membrane depth of retinal in the purple membrane (PM). Chelates of Tb(III) are excellent energy donors for the retinal chromophore of PM, having a maximum Ro value for Förster energy transfer of approximately 62 A (assuming a donor quantum yield of 1). Energy transfer rates were measured from the time-resolved emission kinetics of the donor. The distance of closest approach between chelates and the chromophore was estimated by simulating RDL energy-transfer rate constants according to geometric models of either PM sheets or membrane vesicles. The apparent rate constant for RDL energy transfer between Tb(III)HED3A and retinal in PM sheets is 1.5(+/- 0.1) x 10(6) M-1 s-1, corresponding to a depth of approximately 10 +/- 2 A for the retinal chromophore. Cell envelope vesicles (CEVs) from Halobacterium halobium were studied by using RDL energy transfer to assess the proximity of retinal to either the extracellular or intracellular face of the PM. The estimated depth of retinal from the extravesicular face of the PM is 10 +/- 3 A, based on the RDL energy-transfer rate constant. Energy-transfer levels to retinal in the PM were estimated by an indirect method with energy donors trapped in the inner-aqueous space of CEVs. The rate constants derived for this arrangement are too low to be consistent with the shortest depth of retinal deduced for PM sheets. Thus, the intravesticular face of CEVs, corresponding to the cytoplasmic face of cells, is the more distant surface from the chromophore of bacteriorhodopsin.

Bacteriorhodopsins↗

PubMed2004

Multinuclear NMR studies of single lipid bilayers supported in cylindrical aluminum oxide nanopores.

Lipid bilayers were deposited inside the 0.2 microm pores of anodic aluminum oxide (AAO) filters by extrusion of multilamellar liposomes and their properties studied by 2H, 31P, and 1H solid-state NMR. Only the first bilayer adhered strongly to the inner surface of the pores. Additional layers were washed out easily by a flow of water as demonstrated by 1H magic angle spinning NMR experiments with addition of Pr3+ ions to shift accessible lipid headgroup resonances. A 13 mm diameter Anopore filter of 60 microm thickness oriented approximately 2.5 x 10(-7) mol of lipid as a single bilayer, corresponding to a total membrane area of about 500 cm2. The 2H NMR spectra of chain deuterated POPC are consistent with adsorption of wavy, tubular bilayers to the inner pore surface. By NMR diffusion experiments, we determined the average length of those lipid tubules to be approximately 0.4 microm. There is evidence for a thick water layer between lipid tubules and the pore surface. The ends of tubules are well sealed against the pore such that Pr3+ ions cannot penetrate into the water underneath the bilayers. We successfully trapped poly(ethylene glycol) (PEG) with a molecular weight of 8000 in this water layer. From the quantity of trapped PEG, we calculated an average water layer thickness of 3 nm. Lipid order parameters and motional properties are unperturbed by the solid support, in agreement with existence of a water layer. Such unperturbed, solid supported membranes are ideal for incorporation of membrane-spanning proteins with large intra- and extracellular domains. The experiments suggest the promise of such porous filters as membrane support in biosensors.

Aluminum Oxide↗

PubMed2005

Virion proteins of Kaposi's sarcoma-associated herpesvirus.

The proteins that compose a herpesvirus virion are thought to contain the functional information required for de novo infection, as well as virion assembly and egress. To investigate functional roles of Kaposi's sarcoma-associated herpesvirus (KSHV) virion proteins in viral productive replication and de novo infection, we attempted to identify virion proteins from purified KSHV by a proteomic approach. Extracellular KSHV virions were purified from phorbol-12-tetradecanoate-13-acetate-induced BCBL-1 cells through double-gradient ultracentrifugation, and their component proteins were resolved by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Thirty prominent protein bands were excised and subjected to high-performance liquid chromatography ion trap mass spectrometric analysis. This study led to the identification of 24 virion-associated proteins. These include five capsid proteins, eight envelope glycoproteins, six tegument proteins, and five proteins whose locations in the virions have not yet been defined. Putative tegument proteins encoded by open reading frame 21 (ORF21), ORF33, and ORF45 were characterized and found to be resistant to protease digestion when purified virions were treated with trypsin, confirming that they are located within the virion particles. The ORF64-encoded large tegument protein was found to be associated with capsid but sensitive to protease treatment, suggesting its unique structure and array in KSHV virions. In addition, cellular beta-actin and class II myosin heavy chain type A were found inside KSHV virions and associated with tegument-capsid structure. Identification of KSHV virion proteins makes it possible to study the functional roles of these virion proteins in KSHV replication and pathogenicity.

Amino Acid Sequence↗

PubMed1985

The retention and ultrastructural appearances of various extracellular matrix molecules incorporated into three-dimensional hydrated collagen lattices.

Artificial extracellular matrices composed of collagen, glycosaminoglycans (GAG), proteoglycans (PG), plasma fibronectin (FN), and a hyaluronate-binding protein (HABP) have been prepared that morphologically resemble embryonic extracellular matrices in vivo at the light and electron microscope level. The effect of each of the above matrix molecules on the structure and "self-assembly" of these artificial matrices was delineated. (1) Matrix components assembled in vitro morphologically resemble their counterparts in vivo, for the most part. Scanning and transmission electron microscopy indicate that under our assembly and fixation conditions, collagen forms striated fibrils that are 125 nm in diameter, FN forms 30- to 60-nm granules, chondroitin sulfate proteoglycan (CSPG) forms 27- to 37-nm granules, chondroitin sulfate (CS) assembles into 100- to 250-nm spheres, and hyaluronate (HA) appears either as granular mats when fixed with cetylpyridinium chloride (CPC) or as 1.5- to 3-nm microfibrils when preserved with ruthenium red plus tannic acid. These molecules are known to assume the same configurations in embryonic matrices when the same preservation techniques are used with the exception of FN, which generally forms fibrillar arrays. (2) Addition of various matrix molecules can radically change the appearance of the collage gels. HA greatly expands the volume of the gel and increases the space between collagen fibrils. CSPG at low concentrations (less than 1 mg/ml) and CS at high concentrations (greater than 20 mg/ml) bundle the collagen fibrils into twisted ropes. (3) A variety of assays were used to examine binding between various matrix components and retention of these components in the hydrated collagen lattices. These assays included solid-phase binding assays, negative staining of spread mixtures of matrix components, cryostat sections of unfixed mixtures of matrix components, and retention of radiolabeled matrix molecules in fixed and washed gels. A number of these binding interactions may play a role in the assembly and stabilization of the matrix. (a) HA, CSPG, and FN bind to collagen. CS appears to only weakly bind to collagen, if at all. (b) FN promotes the increased retention of HA, CSPG, and to a very small degrees, CS, in collagen gels. Conversely, the GAG increase the retention of 3H-FN in the gels. Furthermore, FN binds to HA, CS, and CSPG as demonstrated by solid surface binding assays and morphological criteria. The increased retention of GAG and CSPG by the addition of FN may be due to both stabilization of binding to the collagen and trapping of matrix complexes within the gel. (c) HA binds to both CS and CSPG.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗

PubMed1989

Acid phospholipase A1 in liver--a brief survey.

Acid phospholipase A1 activity in liver (rat, human) is predominantly localized in lysosomes. A minor proportion (less than 3% of the total activity) is also present in the Golgi apparatus and the endoplasmic reticulum, presumably due to enzymatically active precursors of the corresponding lysosomal enzyme. Lysosomal phospholipase A1 is the most important enzyme initiating the intralysosomal catabolism of diacylphosphoglycerides. It has been purified 50,600-fold, with a yield of about 26%. The enzyme prefers phosphatidyl-ethanolamine as a substrate, which at 200 microM and pH 4.5, is hydrolysed at a rate of approximately 8.2 U/mg. Lysosomal phospholipase A1 is a glycoprotein of about 29 kDa with an isoelectric point of pH 5.3. Unspecific extralysosomal endogenous inhibitors of this enzyme are pH range, inorganic cations, and various proteins. Divalent cations are more potent inhibitors than monovalent ones. Most endogenous intra- and extracellular proteins inhibit the enzyme, the cationic species exhibiting high inhibitory potencies, glycoproteins only little. Inhibitory proteins act through their binding to the substrate. Lysosomal phospholipase A1 seems to be an important target in drug-induced lipidosis. This lipid storage disease is caused by various cationic amphiphilic drugs that are trapped intralysosomally by protonation. In lysosomes such compounds raise the pH, interact with the polar lipids to be degraded and the lysosomal lipolytic enzymes, such as phospholipase A1. These mechanisms result in impaired intralysosomal phospholipid degradation and hence in intralysosomal phospholipid accumulation.

Humans↗

PubMed1994

Peroxynitrite-mediated oxidation of albumin to the protein-thiyl free radical.

Nitric oxide reacts with superoxide to produce peroxynitrite, which may be an important mediator of oxidant-induced cellular injury. Here we report that peroxynitrite is able to oxidize a protein, bovine serum albumin (BSA), to the corresponding protein-thiyl free radical as demonstrated by electron paramagnetic resonance (EPR)-spin-trapping experiments with both alpha-phenyl-N-tert-butyl nitrone (PBN) and 5,5-dimethyl-1-pyrroline-N-oxide (DMPO). BSA radical adduct yields increased with pH indicating peroxynitrite anion as its main forming agent. Reaction with peroxynitrite may be another aspect of the antioxidant action of albumin in extracellular fluids.

Cyclic N-Oxides↗

PubMed1998

The Drosophila putative kinase linotte (derailed) prevents central brain axons from converging on a newly described interhemispheric ring.

The lio gene encodes a putative receptor tyrosine kinase, with unique motifs both in the extracellular and catalytic domains (Dura, J.-M., Préat, T., Tully, T., 1993. Identification of linotte, a new gene affecting learning and memory in Drosophila melanogaster. J. Neurogenet. 9, 1-14). We show here that a complete deletion of lio activity causes specific structural defects in the adult brain. Gal4 enhancer-trap lines used as cell markers revealed that in lio mutants central brain axons behave as if they were abnormally attracted by the midbrain area. The Lio protein is expressed in third instar larvae in a few cells at the junction of the cerebral hemispheres. These glial cells form a newly described ring structure, showing an invariable fibrous organization. In the wild-type this ring disappears at midpupation. Our results indicate that the Lio putative kinase plays a major role in the modeling of the adult brain by controlling the fate of the transient interhemispheric ring.

Animals↗

PubMed1995

beta-Amyloid peptide-derived, oxygen-dependent free radicals inhibit glutamate uptake in cultured astrocytes: implications for Alzheimer's disease.

beta-Amyloid (A beta), the central constituent of senile plaques in Alzheimer's disease (AD) brains, was shown by us recently to generate free radicals in an oxygen dependent mechanism. A beta-derived free radicals were detected directly using electron paramagnetic resonance (EPR) spin trapping techniques employing the spin trap phenyl-alpha-tert-butylnitrone (PBN). We have extended these studies to investigate the nature of the oxyradicals derived from A beta peptides, and we show that these free radicals are able to inhibit glutamate uptake in cultured astrocytes. An implication of inhibited astrocyte glutamate uptake in brain is increased extracellular levels of glutamate, which is excitotoxic to neurons. These results support the hypothesis that A beta neurotoxicity in AD may be due in part to A beta-derived, oxygen-dependent free radical inhibition of glutamate uptake.

Alzheimer Disease↗

PubMed1990

slit: an extracellular protein necessary for development of midline glia and commissural axon pathways contains both EGF and LRR domains.

The Drosophila slit locus encodes a protein with four regions containing tandem arrays of a 24-amino-acid leucine-rich repeat (LRR) with conserved flanking sequences (flank-LRR-flank surrounding these arrays), followed by two regions with epidermal growth factor (EGF)-like repeats. Each of these motifs has been implicated in protein-protein interactions as part of an extracellular domain in a variety of other proteins. Analysis of slit cDNA clones reveals that as a consequence of alternative splicing, the locus can code for two distinct protein species differing by 11 amino acids at the carboxyl terminus of the last EGF repeat. The existence of a putative signal sequence and the absence of a transmembrane domain suggest that slit is secreted, an observation supported by an analysis of its expression in tissue culture. Examining the expression pattern of slit in the embryo by antibody staining, enhancer trap detection, and in situ hybridization, we demonstrate that the protein is expressed by a subset of glial cells along the midline of the developing central nervous system. Through immunoelectron microscopy, slit can be seen on the commissural axons traversing the glial cells although it is absent from the cell bodies of these neurons, implying that slit is exported by the glia and distributed along the axons. Finally, we demonstrate that a reduction in slit expression results in a disruption of the developing midline cells and the commissural axon pathways. The embryonic localization, mutant phenotype, and homology of slit to both receptor-binding EGF-like ligands and adhesive glycoproteins suggest that it may be involved in interactions between the midline glial cells, their extracellular environment, and the commissural axons that cross the midline.

Alleles↗

PubMed1985

Fluorescence methods for localizing proteinases and proteinase inhibitors in skeletal muscle.

Proteinases and proteinase inhibitors have become suspect in a wide variety of muscle wasting conditions that might be treatable if knowledge of the cellular locale and function of these molecules were known. Fluorescent probes have been useful in the localization of proteinases in muscle samples from human and animal specimens. These include the histochemical localization of proteinases based on the specific fluorescence of hydrolysis product derivatives, but this approach has been limited to the lysosomal proteinases because of the acidic requirements of the trapping reaction of the primary reaction product. Immunohistochemical techniques do not have the same restrictions and a number of lysosomal and nonlysosomal proteinases have been identified in muscle by this means. Unfortunately, they do not yield any information as to the activity of the enzymes. This is an important consideration since the extracellular environment contains a number of proteinase inhibitors, some of which may be internalized by the cell.

Animals↗

PubMed2001

Repacking of the transmembrane domains of P-glycoprotein during the transport ATPase cycle.

P-glycoprotein (P-gp) is an ABC (ATP-binding cassette) transporter, which hydrolyses ATP and extrudes cytotoxic drugs from mammalian cells. P-gp consists of two transmembrane domains (TMDs) that span the membrane multiple times, and two cytoplasmic nucleotide-binding domains (NBDs). We have determined projection structures of P-gp trapped at different steps of the transport cycle and correlated these structures with function. In the absence of nucleotide, an approximately 10 A resolution structure was determined by electron cryo-microscopy of two-dimensional crystals. The TMDs form a chamber within the membrane that appears to be open to the extracellular milieu, and may also be accessible from the lipid phase at the interfaces between the two TMDs. Nucleotide binding causes a repacking of the TMDs and reduction in drug binding affinity. Thus, ATP binding, not hydrolysis, drives the major conformational change associated with solute translocation. A third distinct conformation of the protein was observed in the post-hydrolytic transition state prior to release of ADP/P(i). Biochemical data suggest that these rearrangements may involve rotation of transmembrane alpha-helices. A mechanism for transport is suggested.

4-Chloromercuribenzenesulfonate↗

PubMed1993

Regulation of cell pH by K+/H+ antiport in renal epithelial cells.

Acid-loaded opossum kidney (OK) cells secrete H+ by Na+/H+ exchange and by a Na(+)- and HCO3(-)-independent pathway that has not been fully characterized. We studied the Na(+)-independent component by measuring H+ flux using the pH-sensitive trapped indicator 2',7'-bis(2-carboxyethyl)-5(6)- carboxyfluorescein. Two Na(+)-independent H(+)-transport systems were identified in acid-loaded cells perfused with HCO3(-)-free buffers. The minor component appears to be a conductive pathway for H+, over 90% inhibitable by 5 mM barium. The major component is stimulated by extracellular K+ and was fully active in the presence of barium, amiloride, ouabain, 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid, and bumetanide and in the absence of Cl-. Ammonium inhibited the H+ flux by 72% at 50 mM, and the H+ flux could be accelerated two- to threefold by limited proteolysis of intact cells using kallikrein or papain. In cells pretreated with barium, the K(+)-induced H+ flux caused no change of bis-oxonol fluorescence, suggesting an electroneutral pathway. The H+ flux was a saturable function of extracellular K+ (Michaelis constant 55 mM), and flux reversed when the K+ gradient was reversed. Similarly, the H+ flux was a linear function of the H+ gradient and reversed when the H+ gradient reversed. Evidence for ongoing K(+)-induced H+ flux was also found in nonacidified cells. First, changing perfusate K+ from 5 to 50 mM alkalinized baseline cell pH, an effect not reproduced by barium despite an equivalent depolarizing effect. Second, increasing perfusate K+ from 5 to 50 mM completely eliminated the acidification produced by 1 mM amiloride. We conclude that the OK cell expresses two Na(+)-independent acid-base transport systems. One is a barium-sensitive electrogenic H+ conductance and the other functions as an electroneutral K+/H+ antiporter. The antiporter is capable of H+ extrusion from acid-loaded cells but in normal cells functions in the reverse direction, as an H+ loader. The K+/H+ antiporter appears to be one of the major systems regulation cell pH in these cells, balancing the H+ efflux mediated by Na+/H+ exchange.

Acids↗

PubMed2003

Membrane type I matrix metalloproteinase usurps tumor growth control imposed by the three-dimensional extracellular matrix.

Cancer cells are able to proliferate at accelerated rates within the confines of a three-dimensional (3D) extracellular matrix (ECM) that is rich in type I collagen. The mechanisms used by tumor cells to circumvent endogenous antigrowth signals have yet to be clearly defined. We find that the matrix metalloproteinase, MT1-MMP, confers tumor cells with a distinct 3D growth advantage in vitro and in vivo. The replicative advantage conferred by MT1-MMP requires pericellular proteolysis of the ECM, as proliferation is fully suppressed when tumor cells are suspended in 3D gels of protease-resistant collagen. In the absence of proteolysis, tumor cells embedded in physiologically relevant ECM matrices are trapped in a compact, spherical configuration and unable to undergo changes in cell shape or cytoskeletal reorganization required for 3D growth. These observations identify MT1-MMP as a tumor-derived growth factor that regulates proliferation by controlling cell geometry within the confines of the 3D ECM.

Animals↗

PubMed1999

Identification of an extracellular agent [correction of catalyst] of carbon tetrachloride dehalogenation from Pseudomonas stutzeri strain KC as pyridine-2, 6-bis(thiocarboxylate)

Pseudomonas stutzeri strain KC was originally characterized as having, under iron-limiting conditions, novel carbon tetrachloride (CCl(4)) dehalogenation activity, specifically, a net conversion of CCl(4) to CO(2). The exact pathway and reaction mechanisms are unknown, but chloroform is not an intermediate and thiophosgene and phosgene have been identified as intermediates in trapping experiments. Previous work by others using cell-free preparations has shown that cell-free culture supernatants that have been passed through a low-molecular-weight cutoff membrane can confer rapid CCl(4) transformation ability upon cultures of bacteria which otherwise show little or no reactivity toward CCl(4). We used a cell-free assay system to monitor the complete purification of compounds showing CCl(4) degradation activity elaborated by iron-limited cultures of strain KC. Electrospray tandem mass spectroscopy, NMR spectroscopy, and comparisons with synthetic material have identified pyridine-2,6-bis(thiocarboxylate) as a metabolite of strain KC which has CCl(4) transformation activity in the presence of chemical reductants, e.g., titanium[III] citrate or dithiothreitiol, or actively growing bacterial cultures.

Carbon Dioxide↗

PubMed1992

Calcium depletion reduces the destruction of fibrosarcoma cells in the microvasculature of artificially perfused rat hearts.

Earlier studies have shown that most tumour cells (TCs) injected into the circulation die rapidly. The mechanisms behind this rapid elimination of TCs are not known, but some experimental data suggest that mechanical trauma to the cells in the capillary bed plays a role. In the present investigation 725,000 rat fibrosarcoma cells (250,000/ml) were infused into the coronary microcirculation of isolated and artificially perfused (flow rate approx. 6 ml/min), beating and non-beating (Ca2+ excluded from the perfusate) rat hearts. The analyses included calculations of the number of TCs retained in the myocardium 5 min after start of TC infusion and their distribution within the ventricular wall. In addition, ultrastructural studies were performed to identify possible structural changes of trapped TCs and myocardial tissue. Intact and viable TCs in the effluent perfusate were counted. In beating hearts about 20% of the infused TCs were collected microscopically intact after passage through the heart circulation, and of these cells only 32% were viable (in-flow viability 87-91%). In Ca(2+)-depleted hearts the corresponding figures were 29 and 48%, respectively. The difference in viable cell counts was statistically significant (P less than 0.001). The TCs trapped in the left ventricular wall of the myocardium of beating hearts were mainly found in the subepicardial third of the wall, whereas in non-beating hearts the trapped cells were distributed randomly. The ultrastructural appearance of trapped cells differed between the two groups: 82% of cells trapped in beating hearts showed signs of severe damage, with subcellular vacuolization and plasmalemmal disruption, whereas 65% of cells trapped in Ca(2+)-depleted hearts seemed undamaged with intact cell membranes and cytoplasmic organization. The remaining 35% showed variable subcellular disorganization. The results cannot entirely be explained by mechanical TC damage in the microcirculation due to intracapillary deformation. The observations require additional explanatory mechanisms. One possible important TC damaging mechanism, dependent on extracellular Ca2+ levels, could be endothelial cell release of reactive oxygen species.

Animals↗

PubMed1984

Na+-H+ exchange in gastric glands as measured with a cytoplasmic-trapped, fluorescent pH indicator.

We have used the pH-sensitive, fluorescent, cytoplasmic-trapped dye 2',7'-bis(carboxyethyl)-5(6)-carboxyfluorescein (BCECF) to identify Na+-H+ exchange in gastric glands isolated from rabbit stomachs by high-pressure perfusion and collagenase digestion. The fluorescence of BCECF-loaded glands was calibrated in terms of cytosolic pH (pHc) by permeabilizing the cell membranes and titrating the extracellular solution to different pH values. In one set of experiments in Cl--free solutions, glands were treated with 0.1 mM ouabain for 45 min to increase cellular cytosolic molar sodium ion concentration [( Na+]c) to high levels. Subsequent suspension of these cells in a Na+-free Ringer's solution (to generate [Na+]c greater than [Na+]o) caused cells to acidify rapidly (t1/2 approximately equal to 60 sec) from pHc approximately equal to 7.15 to pHc approximately equal to 6.55. Subsequent addition of 100 mM Na+ or Li+, but not K+, caused cells rapidly to increase pHc (t1/2 approximately equal to 30 sec) toward the control value. These changes of pHc were blocked when ouabain-treated glands had been preequilibrated for 10 min with 1 mM amiloride, and this block was overcome by adding 10 microM monensin (an ionophore that artificially exchanges Na+ for H+). In another set of experiments in Cl--containing Ringer's solution, glands were acid-loaded by treatment with 30 mM NH4Cl for 4 min, followed by washing the NH4Cl from the solutions. Under these conditions, pHc decreased from 7.02 to approximately equal to 6.5; subsequent alkalinization of cells back to control pHc was stimulated by Na+ (t1/2 approximately equal to 60 sec), but not K+, and was inhibited by 1 mM amiloride. This amiloride block also was overcome by further addition of 10 microM monensin. We conclude that gastric glands contain a Na+-H+ exchanger that appears independent of Cl-, not activated by K+, and blocked by 1 mM amiloride. This exchanger is likely localized to the serosal membrane of gland cells. Na+-H+ exchange may play an important role in regulation of pHc in oxyntic and chief cells exposed to high luminal acidity, where back diffusion of H+ into cells may occur at rapid rates.

Amiloride↗

PubMed2000

Drosophila bunched integrates opposing DPP and EGF signals to set the operculum boundary.

The Drosophila BMP homolog DPP can function as a morphogen, inducing multiple cell fates across a developmental field. However, it is unknown how graded levels of extracellular DPP are interpreted to organize a sharp boundary between different fates. Here we show that opposing DPP and EGF signals set the boundary for an ovarian follicle cell fate. First, DPP regulates gene expression in the follicle cells that will create the operculum of the eggshell. DPP induces expression of the enhancer trap reporter A359 and represses expression of bunched, which encodes a protein similar to the mammalian transcription factor TSC-22. Second, DPP signaling indirectly regulates A359 expression in these cells by downregulating expression of bunched. Reduced bunched function restores A359 expression in cells that lack the Smad protein MAD; ectopic expression of BUNCHED suppresses A359 expression in this region. Importantly, reduction of bunched function leads to an expansion of the operculum and loss of the collar at its boundary. Third, EGF signaling upregulates expression of bunched. We previously demonstrated that the bunched expression pattern requires the EGF receptor ligand GURKEN. Here we show that activated EGF receptor is sufficient to induce ectopic bunched expression. Thus, the balance of DPP and EGF signals sets the boundary of bunched expression. We propose that the juxtaposition of cells with high and low BUNCHED activity organizes a sharp boundary for the operculum fate.

Amino Acid Sequence↗