Calcium signalling and calcium transport in bone disease.

Calcium transport and calcium signalling mechanisms in bone cells have, in many cases, been discovered by study of diseases with disordered bone metabolism. Calcium matrix deposition is driven primarily by phosphate production, and disorders in bone deposition include abnormalities in membrane phosphate transport such as in chondrocalcinosis, and defects in phosphate-producing enzymes such as in hypophosphatasia. Matrix removal is driven by acidification, which dissolves the mineral. Disorders in calcium removal from bone matrix by osteoclasts cause osteopetrosis. On the other hand, although bone is central to management of extracellular calcium, bone is not a major calcium sensing organ, although calcium sensing proteins are expressed in both osteoblasts and osteoclasts. Intracellular calcium signals are involved in secondary control including cellular motility and survival, but the relationship of these findings to specific diseases is not clear. Intracellular calcium signals may regulate the balance of cell survival versus proliferation or anabolic functional response as part of signalling cascades that integrate the response to primary signals via cell stretch, estrogen, tyrosine kinase, and tumor necrosis factor receptors.

Inhibition of Amazon deforestation and fire by parks and indigenous lands.

Conservation scientists generally agree that many types of protected areas will be needed to protect tropical forests. But little is known of the comparative performance of inhabited and uninhabited reserves in slowing the most extreme form of forest disturbance: conversion to agriculture. We used satellite-based maps of land cover and fire occurrence in the Brazilian Amazon to compare the performance of large (> 10,000 ha) uninhabited (parks) and inhabited (indigenous lands, extractive reserves, and national forests) reserves. Reserves significantly reduced both deforestation and fire. Deforestation was 1.7 (extractive reserves) to 20 (parks) times higher along the outside versus the inside of the reserve perimeters and fire occurrence was 4 (indigenous lands) to 9 (national forests) times higher. No strong difference in the inhibition of deforestation (p = 0. 11) or fire (p = 0.34) was found between parks and indigenous lands. However, uninhabited reserves tended to be located away from areas of high deforestation and burning rates. In contrast, indigenous lands were often created in response to frontier expansion, and many prevented deforestation completely despite high rates of deforestation along their boundaries. The inhibitory effect of indigenous lands on deforestation was strong after centuries of contact with the national society and was not correlated with indigenous population density. Indigenous lands occupy one-fifth of the Brazilian Amazon-five times the area under protection in parks--and are currently the most important barrier to Amazon deforestation. As the protected-area network expands from 36% to 41% of the Brazilian Amazon over the coming years, the greatest challenge will be successful reserve implementation in high-risk areas of frontier expansion as indigenous lands are strengthened. This success will depend on a broad base of political support.

The Bax pore in liposomes, Biophysics.

The protein BAX of the Bcl-2-family is felt to be one of the two Bcl-2-family proteins that directly participate in the mitochondrial cytochrome c-translocating pore. We have studied the kinetics, stoichiometry and size of the pore formed by BAX in planar lipid bilayers and synthetic liposomes. Our data indicate that a cytochrome c-competent pore can be formed by in-membrane association of BAX monomers.

Intragastric acid control in non-steroidal anti-inflammatory drug users: comparison of esomeprazole, lansoprazole and pantoprazole.

BACKGROUND: Studies to date have not directly compared the pharmacodynamic efficacies of different proton pump inhibitors in controlling intragastric acidity in patients treated with non-steroidal anti-inflammatory drugs. AIM: To compare acid suppression with once-daily esomeprazole 40 mg, lansoprazole 30 mg and pantoprazole 40 mg in patients receiving non-selective or cyclo-oxygenase-2-selective non-steroidal anti-inflammatory drug therapy. METHODS: In this multicentre, open-label, comparative, three-way crossover study, adult patients (n = 90) receiving non-steroidal anti-inflammatory drugs were randomized to one of six treatment sequences. At the study site, patients were administered esomeprazole 40 mg, lansoprazole 30 mg and pantoprazole 40 mg for 5 days each, with a washout period of > or =10 days between each treatment. Twenty-four-hour pH testing was performed on day 5 of each dosing period. RESULTS: The mean percentage of time during the 24-h pH monitoring period that gastric pH was >4.0 was significantly greater with esomeprazole (74.2%) compared with lansoprazole (66.5%; P < 0.001) and pantoprazole (60.8%; P < 0.001), and significantly greater with esomeprazole (P < 0.05) than with the comparators regardless of whether using non-selective vs. cyclo-oxygenase-2-selective non-steroidal anti-inflammatory drugs. CONCLUSIONS: At the doses studied, esomeprazole treatment provides significantly greater gastric acid suppression than lansoprazole or pantoprazole in patients receiving non-selective or cyclo-oxygenase-2-selective non-steroidal anti-inflammatory drugs.

CLIC-1 functions as a chloride channel when expressed and purified from bacteria.

CLIC-1 is a member of a family of proteins related to the bovine intracellular chloride channel p64 which has been proposed to function as a chloride channel. We expressed CLIC-1 as a glutathione S-transferase fusion protein in bacteria. The fusion protein was purified by glutathione affinity, and CLIC-1 was released from its fusion partner by digestion with thrombin. After further purification, CLIC-1 was reconstituted into phospholipid vesicles by detergent dialysis. Chloride permeability of reconstituted vesicles was assessed using a valinomycin dependent chloride efflux assay, demonstrating increased vesicular chloride permeability with CLIC-1 compared with control. CLIC-1-dependent chloride permeability was inhibited by indanyloxyacetic acid-94 with an apparent IC(50) of 8.6 micrometer. The single channel properties of CLIC-1 were determined using the planar lipid bilayer technique. We found that CLIC-1 forms a voltage-dependent, Cl-selective channel with a rectifying current-voltage relationship and single channel conductances of 161 +/- 7.9 and 67.5 +/- 6.9 picosiemens in symmetric 300 and 150 mm KCl, respectively. The anion selectivity of this activity is Br approximately Cl > I. The open probability of CLIC-1 channels in planar bilayers was decreased by indanyloxyacetic acid-94 with an apparent IC(50) of 86 micrometer at 50 mV. These data convincingly demonstrate that CLIC-1 is capable of forming a novel, chloride-selective channel in the absence of other subunits or proteins.

Pro-apoptotic cascade activates BID, which oligomerizes BAK or BAX into pores that result in the release of cytochrome c.

We review data supporting a model in which activated tBID results in an allosteric activation of BAK, inducing its intramembranous oligomerization into a proposed pore for cytochrome c efflux. The BH3 domain of tBID is not required for targeting but remains on the mitochondrial surface where it is required to trigger BAK to release cytochrome c. tBID functions not as a pore-forming protein but as a membrane targeted and concentrated death ligand. tBID induces oligomerization of BAK, and both Bid and Bak knockout mice indicate the importance of this event in the release of cytochrome c. In parallel, the full pro-apoptotic member BAX, which is highly homologous to BAK, rapidly forms pores in liposomes that release intravesicular FITC-cytochrome c approximately 20A. A definable pore progressed from approximately 11A consisting of two BAX molecules to a approximately 22A pore comprised of four BAX molecules, which transported cytochrome c. Thus, an activation cascade of pro-apoptotic proteins from BID to BAK or BAX integrates the pathway from surface death receptors to the irreversible efflux of cytochrome c. Cell Death and Differentiation (2000) 7, 1166 - 1173

Expression and regulation of RAB3 proteins in osteoclasts and their precursors.

The ruffled membrane, the resorptive organelle of the osteoclast, is generated by fusion of intracytoplasmic acidifying vesicles with the plasma membrane, an event analogous to regulated exocytosis. While the ruffled membrane is essential to the bone resorptive process, the mechanisms governing its generation are unknown. However, regulated exocytosis is mediated, in part, by isoforms of the Rab3 subset of Rab GTPases. Because of similarities between exocytosis and ruffled membrane formation, we asked if Rab3 proteins are expressed by osteoclasts or their precursors, and if so, are these molecules regulated by agents known to prompt the osteoclast phenotype? We find murine osteoclast precursors, in the form of bone marrow macrophages (BMMs), express at least two Rab3 isoforms, namely A and B/C, which are individually enhanced by a variety of hematopoietic cytokines. Consistent with the osteoclastogenic properties of a number of these cytokines, differentiation of BMMs into osteoclasts, in vitro, is associated with increased expression of both isoforms, particularly Rab3B/C. Finally, Rab3B/C localizes with the avian osteoclast H+ATPase (vacuolar proton pump) and pp60c-src, both intracellularly and within acidifying vesicles derived largely from the ruffled membrane. Thus, expression of specific rab3 proteins, an event which may control formation of the osteoclast ruffled membrane, is modulated by cytokines during osteoclastogenesis.

Parasitophorous vacuoles of Leishmania mexicana acquire macromolecules from the host cell cytosol via two independent routes.

The intracellular parasite Leishmania survives and proliferates in host macrophages. In this study we show that parasitophorous vacuoles of L. mexicana gain access to cytosolic material via two different routes. (1) Small anionic molecules such as Lucifer Yellow are rapidly transported into the vacuoles by an active transport mechanism that is sensitive to inhibitors of the host cell's organic anion transporter. (2) Larger molecules such as fluorescent dextrans introduced into the host cell cytosol are also delivered to parasitophorous vacuoles. This transport is slower and sensitive to modulators of autophagy. Infected macrophages were examined by two novel assays to visualize and quantify this process. Immunoelectron microscopy of cells loaded with digoxigenin-dextran revealed label in multivesicular endosomes, which appeared to fuse with parasitophorous vacuoles. The inner membranes of the multivesicular vesicles label strongly with antibodies against lysobisphosphatidic acid, suggesting that they represent a point of confluence between the endosomal and autophagosomal pathways. Although the rate of autophagous transfer was comparable in infected and uninfected cells, infected cells retained hydrolyzed cysteine proteinase substrate to a greater degree. These data suggest that L. mexicana-containing vacuoles have access to potential nutrients in the host cell cytosol via at least two independent mechanisms.

Laparoscopic myotomy in achalasia: intraoperative evidence for myotomy of the gastric cardia.

The myotomy performed for achalasia of the esophagus should divide all of the constricting, diseased muscular elements that obstruct the esophagogastric junction (EGJ). Whether the disease process includes proximal gastric as well as esophageal components is as yet unclear, but anatomic evidence complemented by clinical data suggest that the disease process does not end at the evanescent and poorly defined EGJ. Clinical reports from enthusiastic proponents of a particular operative approach for achalasia have not been illuminating in this regard, because all patients are improved to some degree post-operatively, and there are no objective parametric standards for the evaluation of swallowing function. This study reports a series of patients in whom endoscopic viewing was used to judge the adequacy of myotomy after 'esophageal' myotomy. The question posed by this study was, 'Does esophageal myotomy remove all constricting elements at the gastroesophageal junction?' Laparoscopic myotomy was performed in 48 patients with a diagnosis of achalasia; these patients are the most recent in a total cohort of 72 patients operated upon for achalasia during the past 20 years. Myotomy was begun on the esophagus, and extended to the esophagogastric junction; anatomic landmarks, including the appearance of submucosal veins, guided the initial dissection. Intraoperative endoscopy was then performed to determine whether there was residual constriction of the channel between the esophagus and stomach; if so, myotomy was extended onto the gastric cardia until visual evidence of obstruction had disappeared. All patients had either Toupet fundoplication or Dor fundoplication after myotomy. There were obvious constricting elements distal to the gastroesophageal junction in 90% of the patients. These patients required extension of the myotomy onto the stomach for an average of 15 mm. All but one patient had improved swallowing post-operatively. Eight patients required 'stretch' of the distal esophagus/cardia within the first year post-operatively; one patient was reoperated for fibrous scar obstruction of the distal esophagus. Esophageal myotomy limited to the esophageal muscle does not remove all constricting elements at the gastroesophageal junction; as a result, the extended myotomy must be complemented by an antireflux procedure during operations for achalasia.

Ligand binding results in divalent cation displacement from the alpha 2 beta 1 integrin I domain: evidence from terbium luminescence spectroscopy.

The alpha 2 beta 1 integrin serves as a cell surface collagen or collagen/laminin receptor. Binding of the integrin to its ligands is largely mediated by the alpha 2 subunit I domain and requires the presence of divalent cations. Terbium ion (Tb3+), a fluorescent trivalent cation that often binds divalent cation-binding sites on proteins, supported binding of the I domain to collagen with half-maximal binding occurring at 5.2 +/- 1.7 microM Tb3+. By fluorescence resonance energy transfer spectroscopy, Tb3+ showed specific and saturable binding to the recombinant I domain with a Kd of 27 +/- 4 microM. Although both Mg2+ and Mn2+ were capable of quenching Tb3+ fluorescence, Mn2+ was much more effective than Mg2+. The alpha 2 beta 1 integrin also binds the pro-alpha 1(I) collagen carboxyl-terminal propeptide in a Mg2+-dependent manner via the I domain. Recombinant propeptide was used to examine the effect of ligand on the Tb3+ binding properties of the alpha 2 integrin I domain. As propeptide bound to the I domain, Tb3+ fluorescence progressively diminished suggesting that as ligand binds to the I domain, either Tb3+ is displaced or its fluorescence is quenched. Consistent with the former possibility, little dissociation of collagen-bound I domain occurred upon the addition of EDTA and subsequent incubation. These data support a model in which (1) the divalent cation is required for initial ligand-binding activity of the I domain and (2) ligand binding results in subsequent metal ion displacement to generate a metal-free I domain-ligand complex.

Nonesterified fatty acids induce transmembrane monovalent cation flux: host-guest interactions as determinants of fatty acid-induced ion transport.

Nonesterified fatty acids are key intermediates in cellular metabolism whose intracellular concentration is regulated by multiple anabolic, catabolic, and oxidative enzymatic cascades. Herein, we demonstrate that fatty acids induce transmembrane monovalent cation flux with an apparent rate constant kapp = 10(-)4 - 10(-)3 s-1. Fatty acid-induced cation efflux exploits the ionic association of the cation with the carboxylate anion of the fatty acid and the subsequent transmembrane flip-flop of the fatty acid-cation complex. Rates of fatty acid-induced transmembrane cation flux were dependent upon complex host-guest interactions between the fatty acid-cation complex and the phospholipid constituents which comprise the membrane bilayer including (1) the degree of unsaturation of the fatty acid guest and the regiospecificity and stereospecificity of its olefinic linkages; (2) the phospholipid subclass and individual molecular species which constitute the host membrane phospholipids; (3) impedance matching of host and guest hydrophobic characteristics; and (4) the cholesterol content of the membrane bilayer. Arrhenius analysis demonstrated that fatty acid-induced K+ efflux was facilitated largely by changes in the entropy of activation of ion translocation and not the energy of activation. Moreover, Arrhenius analysis demonstrated that the energy of activation of ion translocation was phospholipid subclass specific. For example, arachidonic acid-induced cation efflux in membranes comprised of 16:0-18:1 plasmenylcholine possessed an Ea = 5.3 +/- 0.4 kcal/mol, while that for 16:0-18:1 phosphatidylcholine was 7.2 +/- 0.5 kcal/mol. Electrophysiologic measurements of planar lipid membranes containing 10 mol % arachidonic acid as a substitutional impurity confirmed the ability of physiologically relevant amounts of fatty acid to induce ion translocation with a specific conductance of 2.6 +/- 0.3 microS/cm2. Collectively, these results demonstrate that fatty acids facilitate transmembrane cation flux by an ion carrier type mechanism and suggest that fatty acid-mediated ion transport contributes to the leakage current present in many cell types and thus potentially modulates cellular responsivity during signal transduction where the intracellular content of fatty acids changes dramatically.

Functional expression of p64, an intracellular chloride channel protein.

p64 is a protein identified as a chloride channel by biochemical purification from kidney microsomes. We expressed p64 in HeLa cells using a recombinant vaccinia virus/T7 RNA polymerase driven system. Total cell membranes were prepared from infected/transfected cells and fused to a planar lipid bilayer. A novel chloride channel activity was found in cells expressing p64 and not in control cells. The p64-associated activity shows strong anion over cation selectivity. Single channels show prominent outward rectification with single channel conductance at positive potentials of 42 pS. The chloride channel activity is activated by treatment of the membranes with alkaline phosphatase and inhibited by DNDS and by TS-TM calix(4)arene. Whole membrane anion permeability was determined by a chloride efflux assay, revealing that membranes from cells expressing p64 showed a small but highly significant increase in chloride permeability, consistent with expression of a novel chloride channel activity.

Cytokine activation leads to acidification and increases maturation of Mycobacterium avium-containing phagosomes in murine macrophages.

Mycobacterium avium (MAC) organisms multiply in phagosomes that have restricted fusigenicity with lysosomes, do not acidify due to a paucity of vacuolar proton-ATPases, yet remain accessible to recycling endosomes. During the course of mycobacterial infections, IFN-gamma-mediated activation of host and bystander macrophages is a key mechanism in the regulation of bacterial growth. Here we demonstrate that in keeping with earlier studies, cytokine activation of host macrophages leads to a decrease in MAC viability, demonstrable by bacterial esterase staining with fluorescein diacetate as well as colony-forming unit counts from infected cells. Analysis of the pH of MAC phagosomes demonstrated that the vacuoles in activated macrophages equilibrate to pH 5.2, in contrast to pH 6.3 in resting phagocytes. Biochemical analysis of MAC phagosomes from both resting and activated macrophages confirmed that the lower intraphagosomal pH correlated with an increased accumulation of proton-ATPases. Furthermore, the lower pH is reflected in the transition of MAC phagosomes to a point no longer accessible to transferrin, a marker of the recycling endosomal system. These alterations parallel the coalescence of bacterial vacuoles from individual bacilli in single vacuoles to communal vacuoles with multiple bacilli. These data demonstrate that bacteriostatic and bactericidal activities of activated macrophages are concomitant with alterations in the physiology of the mycobacterial phagosome.

Comparison of the ion channel characteristics of proapoptotic BAX and antiapoptotic BCL-2.

The BCL-2 family of proteins is composed of both pro- and antiapoptotic regulators, although its most critical biochemical functions remain uncertain. The structural similarity between the BCL-XL monomer and several ion-pore-forming bacterial toxins has prompted electrophysiologic studies. Both BAX and BCL-2 insert into KCl-loaded vesicles in a pH-dependent fashion and demonstrate macroscopic ion efflux. Release is maximum at approximately pH 4.0 for both proteins; however, BAX demonstrates a broader pH range of activity. Both purified proteins also insert into planar lipid bilayers at pH 4.0. Single-channel recordings revealed a minimal channel conductance for BAX of 22 pS that evolved to channel currents with at least three subconductance levels. The final, apparently stable BAX channel had a conductance of 0.731 nS at pH 4. 0 that changed to 0.329 nS when shifted to pH 7.0 but remained mildly Cl- selective and predominantly open. When BAX-incorporated lipid vesicles were fused to planar lipid bilayers at pH 7.0, a Cl--selective (PK/PCl = 0.3) 1.5-nS channel displaying mild inward rectification was noted. In contrast, BCL-2 formed mildly K+-selective (PK/PCl = 3.9) channels with a most prominent initial conductance of 80 pS that increased to 1.90 nS. Fusion of BCL-2-incorporated lipid vesicles into planar bilayers at pH 7.0 also revealed mild K+ selectivity (PK/PCl = 2.4) with a maximum conductance of 1.08 nS. BAX and BCL-2 each form channels in artificial membranes that have distinct characteristics including ion selectivity, conductance, voltage dependence, and rectification. Thus, one role of these molecules may include pore activity at selected membrane sites.

Characterization of the osteoclast ruffled border chloride channel and its role in bone resorption.

Bone resorption by osteoclasts requires massive transcellular acid transport, which is accomplished by the parallel action of a V-type proton pump and a chloride channel in the osteoclast ruffled border. We have studied the molecular basis for the appearance of acid transport as avian bone marrow mononuclear cells acquire a bone resorptive phenotype in vitro. We demonstrate a critical role for regulated expression of a ruffled border chloride channel as the cells become competent to resorb bone. Molecular characterization of the chloride channel shows that it is related to the renal microsomal chloride channel, p64. In planar bilayers, the ruffled border channel is a stilbene sulfonate-inhibitable, outwardly rectifying chloride channel. A mechanism by which outward rectification of the single channel chloride current could allow efficient regulation of acidification by the channel is discussed.

Substrate recognition by osteoclast precursors induces C-src/microtubule association.

The osteoclast is distinguished from other macrophage polykaryons by its polarization, a feature induced by substrate recognition. The most striking component of the polarized osteoclast is its ruffled membrane, probably reflecting insertion of intracellular vesicles into the bone apposed plasmalemma. The failure of osteoclasts in c-src-/- osteopetrotic mice to form ruffled membranes indicates pp60(c-src) (c-src) is essential to osteoclast polarization. Interestingly, c-src itself is a vesicular protein that targets the ruffled membrane. This being the case, we hypothesized that matrix recognition by osteoclasts, and their precursors, induces c-src to associate with microtubules that traffic proteins to the cell surface. We find abundant c-src associates with tubulin immunoprecipitated from avian marrow macrophages (osteoclast precursors) maintained in the adherent, but not nonadherent, state. Since the two proteins colocalize only within adherent avian osteoclast-like cells examined by double antibody immunoconfocal microscopy, c-src/tubulin association reflects an authentic intracellular event. C-src/tubulin association is evident within 90 min of cell-substrate recognition, and the event does not reflect increased expression of either protein. In vitro kinase assay demonstrates tubulin-associated c-src is enzymatically active, phosphorylating itself as well as exogenous substrate. The increase in microtubule-associated kinase activity attending adhesion mirrors tubulin-bound c-src and does not reflect enhanced specific activity. The fact that microtubule-dissociating drugs, as well as cold, prevent adherence-induced c-src/tubulin association indicates the protooncogene complexes primarily, if not exclusively, with polymerized tubulin. Association of the two proteins does not depend upon protein tyrosine phosphorylation and is substrate specific, as it is induced by vitronectin and fibronectin but not type 1 collagen. Finally, consistent with cotransport of c-src and the osteoclast vacuolar proton pump to the polarized plasmalemma, the H+-ATPase decorates microtubules in a manner similar to the protooncogene, specifically coimmunoprecipitates with c-src from the osteoclast light Golgi membrane fraction, and is present, with c-src, in preparations enriched with acidifying vesicles reconstituted from the osteoclast ruffled membrane.