bluuebunny/crossref_metadata_2025 huggingface dataset:
Turnup Turndown of Membrane Operation of Membrane Bioreactors
Authors: HOWELL J. A. , ARNOT T. C. , CHUA H. C.
Published: July 2009

Abstract: Membrane bioreactors can be operated with intermittent permeation and continuous aeration. Aeration close to the surface of a submerged membrane helps to maintain a membrane surface that is free from fouling. The conditions under which this occurs depend on the interaction between flux and aeration rate. Increased flux is possible without severe fouling if the aeration rate is increased. Results of performing membrane operation under the dual intermittency of aeration rate and permeation rate, with permeation also interrupted on a regular cycle, are presented. The results show that membrane plants designed for optimal operation at moderate flux can survive effectively with higher flux operation for restricted periods. Designing to account for such effects could reduce overall plant costs.
Intrinsic T Cell Defects in Systemic Autoimmunity
Authors: KONG PHILIP L. , ODEGARD JARED M. , BOUZAHZAH FARIDA , CHOI JIN‐YOUNG , EARDLEY LEAH D. , ZIELINSKI CHRISTINA E. , CRAFT JOSEPH E.
Published: July 2009

Abstract: Systemic lupus erythematosus (SLE) is an autoimmune disease characterized by loss of T cell tolerance to nuclear antigens. Studies in mice and humans have demonstrated that T cells from individuals with lupus are abnormal. Here, we review the known T cell defects in lupus and their possible biochemical nature, genetic causes, and significance for lupus pathogenesis.
Mechanisms Inducing or Controlling CD8+ T Cell Responses against Self‐ or Non‐Self‐Antigens
Authors: ACCAPEZZATO DANIELE , FRANCAVILLA VITTORIO , PROPATO ANTONELLA , PAROLI MARINO , BARNABA VINCENZO
Published: July 2009

Abstract: Cytotoxic T lymphocytes (CTLs) generally recognize antigens en‐dogenously synthesized within the cells and presented in the form of peptides on class I molecules. However, a large body of evidence suggests that dendritic cells (DCs) have the capacity to capture and deliver exogenous antigens into the major histocompatibility complex (MHC) class I processing pathway. In this paper, we discuss this function, defined as cross‐presentation, and how it is directed, particularly in inducing T cell tolerance, and how it requires special activating signals (such as CD40 ligand) to transform into a mechanism that provides either protective immunity or autoimmunity.
The V(D)J Recombination/DNA Repair Factor Artemis Belongs to the Metallo‐β‐Lactamase Family and Constitutes a Critical Developmental Checkpoint of the Lymphoid System
Authors: MOSHOUS DESPINA , CALLEBAUT ISABELLE , DE CHASSEVAL RÉGINA , POINSIGNON CATHERINE , VILLEY ISABELLE , FISCHER ALAIN , DE VILLARTAY JEAN‐PIERRE
Published: July 2009

Abstract:V(D)J recombination constitutes a critical checkpoint in the development of the immune system as shown in several animal models as well as severe combined immune deficiency (SCID) condition in humans. We recently cloned theArtemisgene, whose mutations are responsible for RS‐SCID, a condition characterized by an absence of both B and T lymphocytes and associated with increased sensitivity to ionizing radiations.Artemisis ubiquitously expressed and is localized in the nucleus. Artemis belongs to the metallo‐β‐lactamase superfamily and defines a new group, β‐CASP, within this family. β‐CASP proteins are β‐lactamases acting on nucleic acids. While RS‐SCID patients harborArtemisloss‐of‐function mutations, we identified four patients with a combined immunodeficiency characterized by a low but detectable number of both B and T lymphocytes caused by hypomorphic mutations in theArtemisgene. Two of these patients developed aggressive B cell lymphomas, a condition that suggests Artemis may be considered a ‘caretaker’ factor, similarly to the other V(D)J recombination/DNA repair actors.
Hypermutation in Human B Cells in Vivo and in Vitro
Authors: WELLER SANDRA , FAILI AHMAD , AOUFOUCHI SAID , GUÉRANGER QUENTIN , BRAUN MORITZ , REYNAUD CLAUDE‐AGNÈS , WEILL JEAN‐CLAUDE
Published: July 2009

Abstract: We develop our previous observation that a subpopulation of circulating memory IgM+IgD+CD27+B cells belongs to a separate pathway of differentiation in humans. This subpopulation, which represents 5–25% of peripheral B cells, is also present in spleen in the same proportion and displays a marginal‐zone‐like B cell phenotype. In addition, we describe a short‐time in vitro induction model for somatic hypermutation by using the BL2 Burkitt's lymphoma cell line stimulated by a combination of antibodies directed against different surface receptors. This short‐time assay allows us to show that mutations are stably introduced in one DNA strand of the BL2 VH gene in the G1 phase of the cell cycle.
The Role of relB in Regulating the Adaptive Immune Response
Authors: ZANETTI MAURIZIO , CASTIGLIONI PAOLA , SCHOENBERGER STEPHEN , GERLONI MARA
Published: July 2009

Abstract: Dendritic cells (DCs), which represent a key type of antigen‐presenting cell (APC), are important for the development of innate and adaptive immunity. DCs are involved in T cell activation in at least two main ways: priming via direct processing/presentation of soluble antigen taken up from the microenvironment (conventional priming), and processing/presentation of antigen released from other cells (cross‐priming). relB, a component of the NF‐κB complex of transcription factors, is a critical regulator of the differentiation of DCs. In mice, lack of relB impairs DCs derived from bone marrow both in number and function. Here relB (−/−) bone marrow chimera mice is used to study the APC function of residual DCs in presentation of soluble antigen and cross‐priming. It is found that the DCs in these mice are profoundly deficient in their ability to both prime and cross‐prime T cell responses. It was concluded that the relB gene is involved in regulating the APC function of DCs in vivo.
Tolerogenic Dendritic Cells Induced by Vitamin D Receptor Ligands Enhance Regulatory T Cells Inhibiting Autoimmune Diabetes
Authors: ADORINI LUCIANO
Published: July 2009

Abstract: Dendritic cells (DCs) not only induce but also modulate T cell activation. 1,25‐Dihydroxyvitamin D3 [1,25‐(OH)2D3] induces DCs with a tolerogenic phenotype, characterized by decreased expression of CD40, CD80, and CD86 co‐stimulatory molecules, low IL‐12, and enhanced IL‐10 secretion. We have found that a short treatment with 1,25‐(OH)2D3 induces tolerance to fully mismatched mouse islet allografts, and that this tolerance is stable to challenge with donor‐type spleen cells and allows acceptance of donor‐type vascularized heart grafts. This effect is enhanced by co‐administration of mycophenolate mofetil (MMF), a selective inhibitor of T and B cell proliferation, that also has effects similar to 1,25‐(OH)2D3 on DCs. Graft acceptance is associated with impaired development of type 1 CD4+ and CD8+ cells and an increased percentage of CD4+CD25+ regulatory cells expressing CD152 in the spleen and in the draining lymph node. Transfer of CD4+CD25+ cells from tolerant mice protects 100% of the syngeneic recipients from islet allograft rejection. CD4+CD25+ cells that are able to inhibit the T cell response to a pancreatic autoantigen and to significantly delay disease transfer by pathogenic CD4+CD25− cells are also induced by treatment of adult nonobese diabetic (NOD) mice with a selected vitamin D receptor (VDR) ligand. This treatment arrests progression of insulitis and Th1 cell infiltration, and inhibits diabetes development at non‐hypercalcemic doses. The enhancement of CD4+CD25+ regulatory T cells able to mediate transplantation tolerance and to arrest type 1 diabetes development by a short oral treatment with small organic compounds that induce tolerogenic DCs, like VDR ligands, suggests possible clinical applications of this approach.
The Metaphorical Foundations of Chemical Explanation
Authors: BROWN THEODORE L.
Published: July 2009

Abstract: To address the question of whether typical chemical explanations have characteristics that distinguish them from explanations in other sciences, it is important to recognize the deeply metaphorical nature of scientific explanation in general. The theory of conceptual metaphor, based on results from modern cognitive sciences, postulates that largely unconscious thought processes, grounded in embodied experience and in experiences drawn from the social domain, are at the core of scientific reasoning. I argue, using several examples, that models employed by chemists as explanatory instruments are metaphorical in nature and have a character distinguishable from explanatory representations employed in other fields of science.
How G.N. Lewis Reset the Terms of the Dialogue between Chemistry and Physics
Authors: BOGAARD PAUL A.
Published: July 2009

Abstract: While quantum physics provided a mathematical framework for understanding the valency of chemical elements, to make use of these mathematical equations required approximations based on chemical experience. To better understand how this establishes limits to the presumption that chemistry can be derived from physics, this paper starts with the warnings in Coulson's widely read text, Valence, as to why there are pitfalls in the interpretation of what ‘valence’ accomplishes. He directs some of these concerns back to Pauling's earlier text, The Nature of the Chemical Bond. Both Coulson and Pauling are aware that quantum theory's ability to approximate the energetic stability of chemical systems is of use to chemistry only if it can be correlated with the molecular structure that results from chemical bonding. While this may now be routinely expected, it was a struggle to see that this was the challenge chemistry posed to physics—and this achievement they both attribute to G.N. Lewis. Until the more mature forms of quantum mechanics arose in the 1920s, Lewis was skeptical that the dynamic models of the atom posed by Bohr could meet this challenge. How Lewis formulates this challenge initially in 1916 and then in his longer work, Valence and the Structure of Atoms and Molecules, will be examined.
The Philosophy of Kitaro Nishida and Current Concepts of the Origin of Life
Authors: HOJO K.O.
Published: July 2009

Abstract: The philosophy of Kitaro Nishida is a basic metaphysics that intends to have a perspective beyond both Eastern and Western traditions. Nishida holds that the fundamental reality of the world appears from ‘the place of the absolute nothingness.’ Nishida's thought can be seen to have striking relationships to certain aspects of contemporary science and philosophy.
Constructivism, Relativism, and Chemical Education
Authors: SCERRI ERIC
Published: July 2009

Abstract: Whereas most scientists are highly critical of constructivism and relativism in the context of scientific knowledge acquisition, the dominant school of chemical education researchers appears to support a variety of such positions. By reference to the views of Herron, Spencer, and Bodner, I claim that these authors are philosophically confused, and that they are presenting a damaging and anti‐scientific message to other unsuspecting educators. Part of the problem, as I argue, is a failure to distinguish between pedagogical constructivism regarding students' understanding of science, and constructivism about the way that scientific knowledge is acquired by expert scientists.
Renal Na,K‐ATPase Structure from Cryo‐electron Microscopy of Two‐Dimensional Crystals
Authors: HEBERT HANS , PURHONEN PASI , THOMSEN KAREN , VORUM HENRIK , MAUNSBACH ARVID B.
Published: July 2009

Abstract: The molecular structure of Na,K‐ATPase was determined by electron crystallography from two‐dimensional crystals induced in purified membranes isolated from the outer medulla of pig kidney. The P2 type unit cell contains two protomers in the E2 conformation, each of them with a size of 65 × 75 × 150 Å3. The α, β, and γ subunits in the membrane crystals were demonstrated in the crystals with Western blotting and related to distinct domains in the density map. The α subunit corresponds to most of the density in the transmembrane region as well as to the large hydrophilic headpiece on the cytoplasmic side of the membrane. The headpiece is divided into three separated domains. One of these gives rise to an elongated projection onto the membrane plane, while the putative nucleotide binding and phosphorylation domains form compact densities in the rest of the cytoplasmic part of the structure. Density on the extracellular face corresponds to the protein part of the β subunit. Ten helices from the catalytic a subunit correspond to two groups of distinct densities in the transmembrane region. The structure of the lipid bilayer spanning part also suggests positions for the transmembrane helices from the β and γ subunits. The overall structure of the α subunit of Na,K‐ATPase as determined here by cryo‐electron microscopy is similar to the X‐ray structure of Ca‐ATPase. However, conformational changes between the E1 and E2 forms are suggested by different relative positions of cytoplasmic domains.
Importance of Transmembrane Segment M3 of Na+,K+‐ATPase for Control of Conformational Changes and the Cytoplasmic Entry Pathway for Na+
Authors: VILSEN BENTE , TOUSTRUP‐JENSEN MADS
Published: July 2009

Abstract: A series of mutations were introduced into the sequence Glu282‐Ile‐Glu‐His‐Phe‐Ile‐His288 of the NH2‐terminal part of M3 of the rat kidney Na+,K+‐ATPase, and the resulting mutant pumps were analyzed functionally. Several of the mutations affected the conformational transitions between E1 and E2 forms of dephospho‐ and phosphoenzyme. Mutations to Glu282 and Phe286 affected the E1‐E2 and E1P‐E2P equilibria in parallel, indicating a role for these two residues in both conformational changes. Mutation to His285 preferentially affected the E1P‐E2P equilibrium, and mutation to Ile283 affected only the E1‐E2 equilibrium of the dephosphoenzyme, demonstrating that the conformational changes of M3 in the phospho‐ and dephospho‐forms are not identical. Several of the mutants showed a reduced apparent affinity for Na+, pointing to an important role of the region in optimizing the Na+ binding properties of the enzyme. It is possible that this part of M3 is closely associated with an entry pathway through which the Na+ ions pass from the cytoplasmic surface to reach the cation‐binding pocket. Some of the mutants also displayed an increased Na+‐ATPase activity, and a good correlation was observed between the turnover rate for Na+‐ATPase activity and the rate of dephosphorylation in the absence of K+, indicating an increased ability of Na+ to activate dephosphorylation of E2P by binding in place of K+ at the extracellularly facing sites. Thus, M3 also seems to be a part of the signaling pathway between the external cation sites and the catalytic site.
Mutagenesis of Residues Involved in Control of the Ca2+ Entry Pathway and Conformational Changes Associated with Ca2+ Binding in the SR Ca2+‐ATPase
Authors: ANDERSEN JENS PETER , CLAUSEN JOHANNES D. , EINHOLM ANJA PERNILLE , VILSEN BENTE
Published: July 2009

Abstract: Rapid kinetic measurements were used to study the rate of Ca2+ dissociation from the high‐affinity Ca2+ sites of the dephosphoenzyme (i.e., from the E1Ca2 form toward the cytoplasmic side) as well as the rate of Ca2+ binding with associated conformational changes (E2→E1Ca2 transition) in the wild type and mutants of the sarcoplasmic reticulum Ca2+‐ATPase expressed in mammalian cells. Cluster mutations as well as single mutations in transmembrane segment M3 resulted in conspicuous effects on the rate of Ca2+ migration. Furthermore, mutation of Asp59 in transmembrane segment M1 to arginine exerted a profound effect on Ca2+ interaction. The data demonstrate an important role for M3 residues in control of the Ca2+ entry pathway and provide functional evidence in support of a close relationship between this pathway and the water‐accessible channel leading between transmembrane segments M1 and M3 in the thapsigargin stabilized E2 structure. In addition, rapid kinetic measurements demonstrated that the hydrogen bond network involving Asp813 of loop L6‐7 and Lys758 of M5 is important for the E2→E1Ca2 transition.
Kinetic Investigations of the Mechanism of the Rate‐Determining Step of the Na+,K+‐ATPase Pump Cycle
Authors: CLARKE RONALD J. , HUMPHREY PAUL A. , LÜPFERT CHRISTIAN , APELL HANS‐JÜRGEN , CORNELIUS FLEMMING
Published: July 2009

Abstract: The kinetics of the E2→ E1 conformational change of unphosphorylated Na+,K+‐ATPase from rabbit kidney were investigated via the stopped‐flow technique using the fluorescent label RH421 (pH 7.4, 24°C). The enzyme was preequilibrated in a solution containing 25 mM histidine and 0.1 mM EDTA to initially stabilize the E2 conformation. On mixing enzyme with NaCl alone, tris‐ATP alone, or NaCl and tris‐ATP simultaneously, a fluorescence decrease was observed. The reciprocal relaxation time, 1/τ, of the fluorescent transient was found to increase with increasing NaCl concentration and reached a saturating value in the presence of 1 mM tris‐ATP of 54 (±3) s−1. The experimental behavior could be described by a binding of Na+ to the enzyme in the E2 state with a dissociation constant of 31 (±7) mM, which induces a subsequent rate‐limiting conformational change to the E1 state. Similar behavior, but with a decreased saturating value of 1/τ, was found when NaCl was replaced by choline chloride. Experiments performed with enzyme from shark rectal gland showed similar effects, but with a lower amplitude of the fluorescence change and a higher saturating value of 1/τ for both the NaCl and choline chloride titrations. The results suggest that Na+ ions or salt in general play a regulatory role, similar to ATP, in enhancing the rate of the rate‐limiting E2→ E1 conformational transition by interaction with the E2 state.
Function and Regulation of the Two Major Plant Plasma Membrane H+‐ATPases
Authors: WOLOSZYNSKA MAGDALENA , KANCZEWSKA JUSTYNA , DRABKIN ARTEM , MAUDOUX OLIVIER , DAMBLY STÉPHANIE , BOUTRY MARC
Published: July 2009

Abstract: Plant plasma membrane H+‐ATPases are encoded by a family of about ten genes organized into five subfamilies. Subfamilies I and II contain the most widely and highly expressed genes. In Nicotiana plumbaginifolia, they are represented, respectively, by pma2 (plasma membrane H+‐ATPase) and pma4. When expressed in the yeast Saccharomyces cerevisiae, the two isoforms show different kinetics and are differently regulated by phosphorylation of the penultimate threonine residue and binding of regulatory 14‐3‐3 proteins. To determine if these differences also occurred in plant tissues, we developed an experimental approach allowing the characterization of a single isoform in the plant. When PMA2 bearing a 6‐His tag was expressed under a strong transcription promoter in Nicotiana tabacum BY2 cells, solubilized from microsomal membranes and purified, the penultimate threonine was found to be phosphorylated, thus validating the model.
Functional Properties of the Human Copper‐Transporting ATPase ATP7B (the Wilson's Disease Protein) and Regulation by Metallochaperone Atox1
Authors: LUTSENKO SVETLANA , TSIVKOVSKII RUSLAN , WALKER JOEL M.
Published: July 2009

Abstract: Wilson's disease protein (WNDP) is a copper‐transporting P1‐type ATPase which plays a key role in normal distribution of copper in a number of tissues, particularly in the liver and the brain. Copper has numerous effects on WNDP, altering its structure, activity, and intracellular localization. To better understand the function of this copper‐transporting ATPase and its regulation by copper, we have recently developed the functional expression systems for WNDP and for Atox1, a cytosolic protein that serves as an intracellular donor of copper for WNDP. Here we summarize the results of our experiments on characterization of the enzymatic properties of WNDP and the effects of Atox1 on the WNDP activity.
Heavy Metal Transport CPx‐ATPases from the Thermophile Archaeoglobus fulgidus
Authors: ARGÜELLO JOSÉ M. , MANDAL ATIN K. , MANA‐CAPELLI SEBASTIAN
Published: July 2009

Abstract: PIB‐type ATPases transport diverse heavy metals (Cu+, Ag+, Cu2+. Zn2+, Cd2+, Pb2+, Co2+) across membranes. Toward understanding their mechanisms of metal selectivity, we are studying thermophilic archaeal PIB‐type ATPases. Like other PIB ATPases, these are characterized by the presence of a cation binding CPX sequence in their 6th transmembrane segment and by cytoplasmic N‐terminus metal binding domains (N‐MBDs). CopA and CopB from the thermophile Archaeoglobus fulgidus were cloned and expressed in E. coli. The resulting proteins were purified in a soluble active form. Typical yields were in the order of 3–5 mg of pure protein per liter of bacterial culture. Both enzymes showed maximum activity at 75–85°C. CopA was activated by Ag+>Cu+ while CopB was activated by Cu2+>Ag+>Cu+. The differences in enzyme selectivity can be explained by different consensus sequences in the transmembrane cation binding domain (CopA: CPC, CopB: CPH). Mutagenesis studies show that the cysteines in the transmembrane CPC site of CopA are necessary for enzyme function, while those in the N‐MBD (CXXC), although not essential, are required for maximum enzyme activity. Different from CopA, CopB has a His‐rich N‐MBD. Removal of this domain reduced enzyme activity without affecting enzyme selectivity. These studies show that these enzymes are an excellent system for structural functional studies directed to explain the mechanisms of metal selectivity by PIB ATPases.
Amino Acids in the TM4‐TM5 Loop of Na,K‐ATPase Are Important for Biosynthesis
Authors: JØRGENSEN JESPER R. , HOUGHTON‐LARSEN JENS , JACOBSEN METTE DORPH , PEDERSEN PER AMSTRUP
Published: July 2009

Abstract: The ten‐transmembrane Na,K‐ATPase α‐subunit exposes very few amino acids to the extra membrane space except for an approximately 408 residue‐long loop between transmembrane segments four and five. The present paper focuses on the role of this loop in biosynthesis of functional Na,K‐ATPase. Expression of 39 mutations in this loop to phylogenetically conserved as well as nonconserved residues showed that only two could be expressed at 30°C. By contrast, only five could not be produced in a functional form at 15°C. A detailed analysis showed that a number of these mutants are temperature‐sensitive folding mutants, as they induce the unfolded protein response at 30°C but not at 15°C. We used an algorithm to predict that residues 868ENGFLIPIHLL878 in the L78 loop exposed to the endoplasmic reticulum lumen constitute the most likely BiP binding site. Correct folding of this sequence may be important in the endoplasmic reticulum quality control, as the same loop is responsible for the α‐β‐associations required to leave this compartment. On the basis of the Ca‐ATPase crystal structure and the presented data, we propose a model to account for the role of the TM4‐TM5 loop in Na,K‐ATPase biosynthesis.
FXYD Proteins: New Tissue‐ and Isoform‐Specific Regulators of Na,K‐ATPase
Authors: GEERING KÄTHI , BÉGUIN PASCAL , GARTY HAIM , KARLISH STEVEN , FÜZESI MARIA , HORISBERGER JEAN‐DANIEL , CRAMBERT GILLES
Published: July 2009

Abstract: The recently defined FXYD protein family contains seven members that are small, single‐span membrane proteins characterized by a signature sequence containing an FXYD motif and three other conserved amino acid residues. Until recently, the functional role of FXYD proteins was largely unknown, with the exception of the γ subunit of Na,K‐ATPase, which was shown to be a specific regulator of renal α1‐β1 isozymes. We have investigated whether other members of the FXYD family may have a similar role as the γ subunit and have found that CHIF (corticosteroid hormone‐induced factor, FXYD4), FXYD7, as well as phospholemman (FXYD1) specifically associate with Na,K‐ATPase and preferentially with α1‐β isozymes in native tissues, and produce distinct effects on the transport properties of Na,K‐ATPase that are adapted to the physiological demands of the tissues in which they are expressed. These results provide evidence for a unique and novel mode of regulation of Na,K‐ATPase by FXYD proteins that involves a tissue‐specific expression of an auxiliary subunit of distinct Na,K‐ATPase isozymes.
Immunocytochemical Localization of Na,K‐ATPase Gamma Subunit and CHIF in Inner Medulla of Rat Kidney
Authors: PIHAKASKI‐MAUNSBACH KAARINA , VORUM HENRIK , LØCKE ELSE‐MERETE , GARTY HAIM , KARLISH STEVEN J. D. , MAUNSBACH ARVID B.
Published: July 2009

Abstract: The γ subunit of Na,K‐ATPase and CHIF both belong to the FXYD single‐membrane‐spanning protein family and have been suggested to have regulatory functions in kidney tubules. CHIF is known to be present in the collecting duct, and γ has been demonstrated in several segments of the rat kidney tubule, but never clearly in the inner medullary collecting duct (IMCD). Here, we demonstrate the cellular and subcellular localization of the γ subunit and CHIF in the IMCD in inner medulla by using Western blotting, laser‐scanning confocal immunofluorescence, and immunoelectron microscopy. In the initial quarter of the IMCD (next to the outer medulla), antibodies against the C‐terminal of γ as well as splice variant γa labeled the basolateral surface of intercalated cells (ICs), while principal cells (PCs) remained unlabeled. In the middle segment of the IMCD, all PCs exhibited distinct basolateral staining for the γC‐terminal as well as γa and CHIF. Immunoelectron microscopy showed that the γC‐terminal and CHIF were associated with the inner leaflet of the basolateral plasma membrane in the labeled cells. Immunoblotting demonstrated the presence of both the γC‐terminal and γa in inner medullary tissue. However, splice variant γb was not detected in inner medulla by immunocytochemistry or immunoblotting. The present observations demonstrate that the Na,K‐ATPase γ subunit and CHIF are strategically located in the inner medulla to participate in the fine‐tuning of urine ion composition through the regulation of the Na,K‐ATPase activity in the IMCD.
Structure/Function Studies of the Gamma Subunit of the Na,K‐ATPase
Authors: BLOSTEIN RHODA , PU HELEN X. , SCANZANO ROSEMARIE , ZOUZOULAS ATHINA
Published: July 2009

Abstract: The Na,K‐ATPase γ subunit is present primarily in kidney as two splice variants, γa and γb, which differ only at their extracellular N‐termini. Two distinct effects of gamma are seen in biochemical Na,K‐ATPase assays of mammalian (HeLa) cells transfected with γa or γb, namely, (i) a decrease in K′ATP probably secondary to a shift in steady‐state E1↔ E2 poise in favor of E1 and (ii) an increase in cytoplasmic K+/Na+ antagonism seen as an increase in K′Na at high K+ concentration. Mutagenesis experiments involving alterations in extramembranous regions of γ indicate that different regions mediate the aforementioned distinct effects and that the effects appear to be long range. Studies of ouabain‐sensitive fluxes with intact cells confirm the γ effects seen with membranes and also suggest an additional effect (increase) in apparent affinity for extracellular K+. Alteration in gamma function was also evidenced in the behavior of a G41 →R mutation within the transmembrane domain of gamma. G41R is associated with autosomal dominant renal magnesium wasting. Our studies show that this mutation in the γb variant retards trafficking of γ, but not αβ pumps, to the cell surface and abolishes functional effects of γ, consistent with the conclusion that the Mg2+ transport defect is secondary to loss of γ modulation of Na,K‐ATPase function.
Characterization of PISP, a Novel Single‐PDZ Protein That Binds to All Plasma Membrane Ca2+‐ATPase b‐Splice Variants
Authors: GOELLNER GEOFFREY M. , DEMARCO STEVEN J. , STREHLER EMANUEL E.
Published: July 2009

Abstract: Plasma membrane Ca2+ ATPases (PMCAs) maintain intracellular Ca2+ homeostasis and participate in the local regulation of Ca2+ signaling. Spatially separate demands for Ca2+ regulation require proper membrane targeting of PMCAs, but the mechanism of PMCA targeting is unknown. Using the PMCA2b carboxyl‐terminal tail as yeast two‐hybrid bait, we isolated a novel PDZ domain‐containing protein from a human brain cDNA library. This protein, named PISP for PMCA‐interacting single‐PDZ protein, consists of 140 amino acids and contains little else besides a single PDZ domain. Pulldown experiments showed that PISP interacts with all PMCA b‐splice forms. PISP was found to be ubiquitously expressed and, in MDCK cells, was present in a punctate pattern throughout the cytosol and at the basolateral membrane. When added to microsomal membranes expressing PMCA4b, PISP was unable to stimulate the PMCA‐dependent ATPase activity. Our data suggest that PISP is a transiently interacting partner of the PMCA b‐splice forms that may play a role in their sorting to or from the plasma membrane.
Na,K‐ATPase as a Signal Transducer
Authors: AIZMAN OLEG , APERIA ANITA
Published: July 2009

Abstract: Recent studies have indicated that Na,K‐ATPase may, in addition to being the key regulator of intracellular Na+ and K+ concentration, act as a signal transducer. Despite extensive research, the biological role for ouabain, a natural ligand of Na,K‐ATPase, is not well understood. We have reported that exposure of rat proximal tubular cells (RPTC) to doses of ouabain that inhibit the Na,K‐ATPase activity by less than 50% (10 nM ‐ 500 μM), will induce intracellular [Ca2+]i oscillations and that this calcium signal leads to activation of the transcription factor NF‐κB. The ouabain‐induced calcium oscillations were blocked by an inhibitor of the IP3 receptors but not by phospholipase C inhibitors nor by cellular depletion of IP3, suggesting that the calcium signal is not due to phospholipase C‐mediated IP3 release. Fluorescence resonance energy transfer (FRET) studies suggested a close proximity between the Na,K‐ATPase and IP3 receptor. Our findings demonstrate a novel principle for calcium signaling via Na,K‐ATPase.
Molecular Mechanisms of Na/K‐ATPase‐Mediated Signal Transduction
Authors: XIE ZIJIAN
Published: July 2009

Abstract: Our recent work shows that in addition to pumping ions, Na/K‐ATPase acts as a signal transducer. Binding of ouabain to Na/K‐ATPase changes the interaction of the enzyme with neighboring membrane proteins and induces the formation of multiple signaling modules, resulting in activation of Src, transactivation of the EGF receptor (EGFR), and increased production of reactive oxygen species (ROS). Interaction of these signals leads to activation of several other cascades, including p42/44 and p38 MAPKs, phospholipase C, and protein kinase C isozymes, in a cell‐specific manner. Ouabain also increases [Ca2+]i and contractility, induces some of the early‐response protooncogenes, and activates transcription factors AP‐1 and NF‐κB. Interplay among these pathways eventually results in changes in the expression of a number of growth‐related genes and in cell growth. Significantly, inhibition of Src blocked many of the aforementioned ouabain‐activated signaling pathways. Furthermore, Src binds to Na/K‐ATPase directly and ouabain regulates the interaction between Src and the enzyme, resulting in Src activation. To address the possibility that the signaling Na/K‐ATPase is concentrated in a separate pool on the plasma membrane, we have assessed interaction of the enzyme with caveolins. These studies indicated that Na/K‐ATPase was concentrated in caveolae/rafts. In addition, caveolin‐1 can be co‐immunoprecipitated with Na/K‐ATPase. Finally, we have shown that the signaling function of the enzyme is also pivotal to ouabain‐induced nongenomic effects on cardiac myocytes.
Low Concentrations of Ouabain Activate Vascular Smooth Muscle Cell Proliferation
Authors: ALLEN JULIUS C. , ABRAMOWITZ JOEL , KOKSOY ASLIHAN
Published: July 2009

Abstract: In vascular smooth muscle cells the sodium pump complex can act as an intracellular signal transducing complex activated by low ouabain concentrations, which inhibit sufficient pumps to activate a transduction cascade via transactivation of EGFR, but insufficient pumps to alter intracellular ions. Higher concentrations interfere with proliferation. This biphasic ouabain response occurs in human, canine, and rat VSMC at concentrations that reflect the differing ouabain affinities of the α1 isoforms of the three species. This supports the proposal that this effect occurs via ouabain binding to the α1 subunit of the Na pump. These data suggest a new transducing function of ouabain‐Na pump interaction, distinct from the cellular ionic effects resulting from pump inhibition. This transducing function occurs at ouabain concentrations that do not perturb cytoplasmic ion content and requires specific localization of pumps to caveolae.
Renal Tubule Sodium Transporter Abundance Profiling in Rat Kidney
Authors: KNEPPER MARK A. , KIM GHEUN‐HO , MASILAMANI SHYAMA
Published: July 2009

Abstract: Based on extensive physiological study of sodium transport mechanisms along the renal tubule, complementary DNAs for all of the major transporters and channels responsible for renal tubular sodium reabsorption have been cloned over the past decade. There is now a comprehensive set of cDNA and antibody probes that can be used to investigate physiological mechanisms on a molecular level. Using rabbit polyclonal antibodies to all of the major renal Na transport proteins, we have developed profiling methods allowing comprehensive, integrated analysis of sodium transporter protein abundance changes along the renal tubule in response to physiological and pathophysiological perturbations. Here, we review some of our recent findings with this approach, focusing on renal responses to aldosterone and to variations in NaCl intake.
Isoform‐Specific Regulation of Na+,K+‐ATPase Endocytosis and Recruitment to the Plasma Membrane
Authors: TEIXEIRA VERA LUCAS , KATZ ADRIAN I. , PEDEMONTE CARLOS H. , BERTORELLO ALEJANDRO M.
Published: July 2009

Abstract: The Na+,K+‐ATPase traffics between the plasma membrane and intracellular compartments in response to acute changes in membrane receptor activation. These effects are accomplished by a time‐dependent interaction of the Na+,K+‐ATPase α‐subunit with specific intracellular signaling molecules either at the plasma membrane (endocytosis) or at the endosome's membranes (recruitment). Most of these studies have been performed in rat renal epithelial cells in which only the α1 isoenzyme is present. Studies in neurons from the neostriatum in which all three α‐subunit isoforms are present indicate that neurotransmitter‐dependent regulation of Na+,K+‐ATPase activity displays isoform specificity and also suggest a more complex organization of the intracellular signaling networks controlling Na+,K+‐ATPase traffic in mammalian cells.
The Sodium Pump Keeps Us Going
Authors: CLAUSEN TORBEN
Published: July 2009

Abstract: This invited lecture reviews recent evidence that, in skeletal muscle, excitability and contractility depend on the transmembrane distribution of Na+ and K+ and the membrane potential, which in turn are determined by the operation of the Na+‐K+ pump. Action potentials are elicited by passive fluxes of Na+ and K+. Because of their size and sudden onset, these transport events constitute the major challenge for the Na+‐K+ pumps. When the Na+‐K+ pumps cannot readily restore the Na+‐K+ gradients, working muscle cells often undergo net loss of K+ and gain of Na+. This leads to loss of excitability and force, in particular, in muscles where excitation‐induced passive Na+‐K+ fluxes are large. Thus, excitability depends on the leak/pump ratio for Na+ and K+. When this ratio is increased by inhibition or downregulation of the Na+‐K+ pumps, the force decline seen during continued stimulation is accelerated. This effect is highly significant already within the first seconds of electrical stimulation. Fortunately, electrical stimulation also increases Na+‐K+ pumping rate within seconds. Thus, maximum increase (20‐fold above the resting level) may be reached in 10 seconds, with utilization of all available Na+‐K+ pumps. In muscles, where excitability was inhibited by exposure to high [K+]o (10‐12.5 mM), activation of the Na+‐K+ pumps by hormones or electrical stimulation restored excitability and contractile force. In working muscles, the Na+‐K+ pumps, because of rapid activation of their large transport capacity, play a dynamic regulatory role in the second‐to‐second ongoing restoration and maintenance of excitability and force. The Na+‐K+ pumps become a limiting factor for contractile endurance, in particular, if their capacity is reduced by inactivity or disease.
Mechanisms of Pressure Natriuresis
Authors: McDONOUGH ALICIA A. , LEONG PATRICK K. K. , YANG LI E.
Published: July 2009

Abstract: An acute increase in blood pressure provokes a rapid decrease in proximal tubule salt and water reabsorption that is central to tubuloglomerular feedback regulation of renal blood flow and glomerular filtration rate and contributes to pressure natriuresis. The molecular mechanisms responsible for this critical homeostatic adjustment were studied. When blood pressure is acutely elevated, apical proximal tubule NHE3 are rapidly redistributed out of the microvilli to intermicrovillar clefts and then endosomal pools, and Na,K‐ATPase activity is suppressed. Depressing apical Na+ entry without hypertension is not sufficient to decrease Na,K‐ATPase activity, and depressing Na,K‐ATPase activity alone is not sufficient to decrease proximal tubule Na+ and water reabsorption; thus, it appears that coordinated decreases in both NHE3 surface distribution and Na,K‐ATPase activity may be important for the response to acute hypertension. Clamping plasma angiotensin II levels blunts the retraction of NHE3 from the cell surface to endosomal pools. The increased volume flow of salt and water to the loop of Henle stimulates Na,K‐ATPase activity in this region and provides evidence for a downstream shift in sodium transport during acute hypertension. These same responses in the proximal tubule and loop develop and persist in the spontaneously hypertensive rat. These studies demonstrate that sodium transporters along the nephron are very dynamic, responding quickly to normal fluctuations of blood pressure, and are key to generating the macula densa tubuloglomerular feedback signal and for accommodating increased volume flow through the loop of Henle.
Ouabain as a Mammalian Hormone
Authors: SCHONER WILHELM , BAUER NATALI , MÜLLER‐EHMSEN JOCHEN , KRÄMER ULRIKE , HAMBARCHIAN NJDE , SCHWINGER ROBERT , MOELLER HANS , KOST HOLGER , WEITKAMP CHRISTINE , SCHWEITZER THOMAS , KIRCH ULRIKE , NEU HORST , GRÜNBAUM ERNST‐GÜNTHER
Published: July 2009

Abstract: Endogenous ouabain changes rapidly in humans and dogs upon physical exercise and is under the control of epinephrine and angiotensin II. Hence, the steroid acts as a rapidly acting hormone. A search for a specific binding globulin for cardiac glycosides in bovine plasma resulted in the identification of the d allotype of the μ chain of IgM whose hydrophobic surfaces interact with cardiotonic steroids and cholesterol. Such IgM complexes might be involved in the hepatic elimination of cardiotonic steroids. Thus, differences in the signaling cascade starting at Na+,K+‐ATPase must explain any differences in the action of ouabain and digoxin in the genesis of arterial hypertension.
Ehrlichiae and Ehrlichial Diseases in China
Authors: WEN BOHAI , CAO WUCHUN , PAN HUA
Published: July 2009

Abstract: The various ticks collected from different areas of China were examined for the existence of ehrlichial agents by polymerase chain reaction (PCR) with genus‐ or species‐specific primers designed on the basis of ehrlichial 16S rRNA genes and sequence analyses. In southern China, E. chaffeensis was detected in Amblyomma testudinarium ticks from infested cattle, Haemaphysalis yeni ticks from hare, and Ixodes ovatus ticks from Muntiacus reevesi. E. canis was identified in Rhipicephalus sanguineus ticks from dogs and Boophilus microplus ticks from goats. A new species of the genus Ehrlichia, closely related to E. chaffeensis, and Anaplasma marginale were found in B. microplus ticks from cattle in Tibet. In northern China, E. chaffeensis was detected in Dermacentor silvarum and I. persulcatus ticks; the granulocytic ehrlichial agents were detected in I. persulcatus ticks from an area where Lyme disease is endemic. Canine ehrlichiosis was found in southern China and E. canis and E. platys were identified in dogs; human ehrlichioses were demonstrated by amplifying the 16S rRNA genes of E. chaffeensis and granulocytic ehrlichial agents from patients' blood specimens. In comparison of 16S rRNA gene sequences, the sequences of E. chaffeensis, E. canis, and E. platys in China were found to be different from that in other countries at certain nucleotide positions. These results reveal that a variety of tick‐borne ehrlichial agents and diseases exist in China, and the ehrlichial agents and their tick‐vectors are same as or different from that in other countries at species or strain levels.
Rickettsia honei
Authors: GRAVES STEPHEN , STENOS JOHN
Published: July 2009

Abstract: Rickettsia honei (also known as strain TT‐118) has been detected on three continents. Originally isolated in Thailand in 1962 (and confirmed in 2001), it has also been detected on Flinders Island (Australia) in 1993 and in Texas (USA) in 1998. On each continent it has been associated with a different species of tick. The original isolate (Thai Tick Typhus strain TT‐118) was from a pool of larval Ixodes and Rhipicephalus ticks. Later it was detected in I. granulatus from Rattus rattus. Its pathogenicity for humans has not yet been confirmed in Thailand, but it is possibly responsible for the Spotted Fever Group human rickettsiosis in Thailand. The strain from Texas (USA) was isolated from Amblyomma cajennense ticks taken from cattle. Its pathogenicity for humans has not yet been confirmed in Texas. However, this tick is known to bite humans. The strain from Flinders Island (Australia) described as R. honei, has been isolated from patients with ‘Flinders Island Spotted Fever’ and from Aponomma hydrosauri ticks taken from blue‐tongue lizards (Tiliqua nigrolutea), tiger snakes (Notechis ater humphreysi), and copperhead snakes (Austrelaps superbus) on Flinders Island. The ecology of R. honei in this location is unusual in that reptiles, rather than mammals, are the vertebrate hosts.
Ultrastructural and Genetic Evidence of a Reptilian Tick, Aponomma hydrosauri, as a Host of Rickettsia honei in Australia
Authors: WHITWORTH TED , POPOV VSEVOLOD , HAN VIOLET , BOUYER DONALD , STENOS JOHN , GRAVES STEPHEN , NDIP LUCY , WALKER DAVID
Published: July 2009

Abstract: In 1993, a novel rickettsia was isolated from the blood of inhabitants of Flinders Island, Australia, with acute febrile illnesses. This rickettsia was found to be a new species of spotted fever group (SFG) rickettsia, eventually named Rickettsia honei. The suspected ectoparasite vector of this rickettsia has yet to be identified. The purpose of this study was to evaluate the presence of this rickettsial species in a suspected tick vector, Aponomma hydrosauri, by DNA sequencing and electron microscopy (EM). Ticks collected from an Australian blue‐tongued lizard on Flinders Island and a copperhead snake in Tasmania were demonstrated to be infected with R. honei by PCR, DNA sequencing, and EM. Rickettsiae were found in ultrathin sections of salivary glands, malpighian tubules, and midgut epithelial cells. In a previous study with a R. honei‐infected tick from Flinders Island, rickettsiae were found in the nuclei of midgut epithelial cells, and EM also revealed the presence of rickettsiae in the cytosol of oocytes and immature eggs, suggesting transovarial transmission. These results implicate A. hydrosauri as a possible host of R. honei on Flinders Island and Tasmania and also provide evidence favoring transovarial maintenance of R. honei.
Candidatus Ehrlichia Walkerii
Authors: BROUQUI P. , SANOGO Y. O. , CARUSO G. , MEROLA F. , RAOULT D.
Published: July 2009

Abstract: Ixodes ricinus harbors many infectious agents pathogenic for humans. A cause of fever is found in less than 50% of patients exposed to ticks. Investigations on 359 Ixodes ticks removed from asymptomatic patients in Northern Italy revealed the presence of a new ehrlichial agent in 10 ticks. Comparison of the 16S rRNA and the gltA gene sequences showed the organism is most closely related to Ehrlichia ruminantium. We propose this new Ehrlichia be named ‘candidatus Ehrlichia walkerii.’
‘Candidatus Rickettsia tarasevichiae’ in Ixodes persulcatus Ticks Collected in Russia
Authors: SHPYNOV STANISLAV , FOURNIER PIERRE‐EDOUARD , RUDAKOV NIKOLAY , RAOULT DIDIER
Published: July 2009

Abstract: We collected 209 Ixodes persulcatus ticks in various regions of Russia, including the southern Urals and western and eastern Siberia. Using PCR amplification and sequencing of the citrate synthase‐encoding gene (gltA), we detected a new rickettsial genotype, which we named ‘Candidatus Rickettsia tarasevichiae.’ This bacterium was found in 9.27%, 10.0%, and 20.5% of the ticks collected in western Siberia, eastern Siberia, and the southern Urals, respectively. ‘Candidatus Rickettsia tarasevichiae’ exhibited a 98% and 96% nucleotide sequence homology, with the 16S rDNA and gltA sequence, respectively, of R. canadensis, a rickettsia previously only found in Haemaphysalis leporispalustris ticks in North America. The phylogenetic analysis of ‘Candidatus Rickettsia tarasevichiae’ and other Rickettsia species allowed the creation of a new cluster with high bootstrap values within the Rickettsia genus involving this rickettsia, R. canadensis, and three uncultured rickettsiae from plant insects.
Identification of Rickettsia spp. and Bartonella spp. in Fleas from the Thai‐Myanmar Border
Authors: PAROLA P. , SANOGO O. Y. , LERDTHUSNEE K. , ZEAITER Z. , CHAUVANCY G. , GONZALEZ J. P. , MILLER R. S. , TELFORD S. R. , WONGSRICHANALAI C. , RAOULT D.
Published: July 2009

Abstract: During a survey for possible rickettsial vectors in villages of the central part of the Thai‐Myanmar border from September 2001 to February 2002, four species of fleas were collected from common peridomestic animals. All fleas were tested by PCR to detect DNA of bacteria of the genera Rickettsia (gltA and ompB genes) and Bartonella (ITS and ftsZ genes). Sequencing of PCR‐amplified products was done using gltA fragments for Rickettsia and ftsZ fragments for Bartonella. Two genotypes related to Rickettsia felis were identified in three Ctenocephalides canis and one C. felis specimen. Further, the following Bartonella spp. were detected: Bartonella henselae in two C. felis specimens; Bartonella clarridgeiae in three C. felis specimens; and a new Bartonella genotype in one Nosopsylla fasciatus specimen. Rickettsia and Bartonella may be frequently detected in fleas infesting peridomestic animals from the western border of Thailand.
Rapid Deployment and Operation of a Q Fever Field Laboratory in Bosnia and Herzegovina
Authors: NICHOLSON WILLIAM L. , McQUISTON JENNIFER , VANNIEUWENHOVEN TY J. , MORGAN EARL W.
Published: July 2009

Abstract: In May 2000, CDC was asked to assemble a team to investigate reports of a widespread outbreak of Q fever (Coxiella burnetii infection) in Bosnia‐Herzegovina. Brucellosis was also suspected in the outbreak. In a short period of time, we were able to gather supplies, establish collaboration, and rapidly deploy a field laboratory for the serodiagnostic testing of animal and human specimens. Strategy, problems, and insights on organization, transport, and deployment will be discussed.
Human Granulocytic Ehrlichiosis in Italy
Authors: RUSCIO MAURIZIO , CINCO MARINA
Published: July 2009

Abstract: We report here the two first cases of human ehrlichiosis described in Italy. One patient presented symptoms indicative of ehrlichiosis, such as fever, conjunctivitis, and increase of hepatic enzymes; HGE was confirmed by fourfold seroconversion for HGE antigen. The second patient, a forest worker, was affected by Lyme disease, and in this case HGE was suspected because of fever, leukopenia, and seroconversion.