bluuebunny/crossref_metadata_2025 huggingface dataset:
Epigenetics and the Environment
Authors: SUTHERLAND JESSICA E. , COSTA MAX
Published: July 2009

Abstract: DNA methylation and histone modification promote changes in chromatin structure that may affect gene expression in a heritable manner without directly altering the genome. As such, these phenomena are considered to be epigenetic in nature and are believed to contribute to the normal processes of human development but also to aberrant disease states such as cancer. Epigenetic processes probably contribute mechanistically to toxicant‐induced changes in gene expression and cancer. Nickel is a potent human carcinogen that has been shown to alter DNA methylation patterns and affect histone acetylation status. Both of these changes are associated with the proximity of the affected regions to heterochromatin. The two processes probably occur in concert in mammalian cells. However, in yeast cells, DNA methylation is absent, and nickel is capable of regulating gene expression through changes in acetylation of the lysine residues in the N terminal tail of histone H4. Arsenic is another important environmental carcinogen, and it is methylated during its metabolism. Hence, it has been proposed that arsenic metabolism may deplete intracellular methyl group stores and thereby lead to changes in DNA methylation that may be involved in carcinogenesis. However, the data concerning DNA methylation changes following arsenic exposure are equivocal, leading researchers to propose that DNA hypo‐ and hypermethylation are both important in the development of arsenic‐induced cancers. Heightened awareness by toxicologists of the importance of epigenetics in normal human development and in carcinogenesis should lead to the identification of other toxicants that manifest their effects, at least in part, via epigenetic mechanisms.
DNA Methylation as a Cancer‐Specific Biomarker
Authors: PATEL ARTI , GROOPMAN JOHN D. , UMAR ASAD
Published: July 2009

Abstract: Cancer contributes to a large proportion of the mortality and morbidity in the United States and worldwide. Despite advances in diagnosis and treatment of various cancers, early detection and treatment of cancer remain a challenge. Diagnosis of cancer often occurs once the disease has progressed to a point where currently available intervention options provide limited success. Therefore, techniques that enable early detection followed by targeted interventions would influence stage at diagnosis and, in turn, mortality associated with cancer. Identification of molecular biomarkers, especially those that are associated with cancer initiation and progression, shows promise as an effective strategy in this regard. One potential early detection biomarker is DNA methylation of the promoter region of certain cancer‐associated genes, which results in gene inactivation. Examination of serum for circulating tumor DNA with abnormal methylation patterns offers a possible method for early detection of several cancers and serves as a point for early intervention and prevention strategies. Additionally, it is imperative to consider how such a screening mechanism can be implemented in populations at risk, especially in resource‐poor settings. Thus, the challenge is to validate DNA methylation as a cancer‐specific biomarker, with the ultimate goal of designing a research plan that integrates the current knowledge base regarding cancer detection and diagnosis into specific prevention and intervention strategies that can be applied at a population level.
Membrane Contactors in the Beverage Industry for Controlling the Water Gas Composition
Authors: CRISCUOLI ALESSANDRA , DRIOLI ENRICO , MORETTI UGO
Published: July 2009

Abstract: In the work described here, membrane contactors are used for coupling the removal of species (oxygen and hydrogen sulfide) present in the water with the water carbonation process. We include both experiments and a theoretical study devoted to the analysis of the transport phenomena that occur in the membrane contactor. The main resistance to mass transport was located at the liquid side. Correlations between Sherwood and Reynolds numbers on the shell side that are suitable for the membrane contactor used to carry out our experiments have been determined. In particular, for Re > 1.6, the expression proposed by Yang and Cussler in 1986: Sh= 0.90Re0.40Sc0.33 describes the behavior of the system; whereas, for Re between 0.03 and 0.3, a new expression is proposed: Sh= 0.435Re1.2Sc0.33. A comparison with traditional equipment is also furnished. Membrane contactors offer reduced size, CO2 consumption, and capital costs.
Membrane Contactors for Textile Wastewater Ozonation
Authors: CIARDELLI GIANLUCA , CIABATTI INGRID , RANIERI LAURA , CAPANNELLI GUSTAVO , BOTTINO ALDO
Published: July 2009

Abstract: This paper deals with the application of a membrane contactor for the ozone treatment of textile wastewater. Ceramic (α‐Al2O3) membranes were chosen because of their ozone resistance. A thin metal oxide (TiO2 and γ‐Al2O3) layer was deposited on the membrane surface to eliminate large defects. Membranes were characterized by bubble pressure and gas permeability tests. Mass transfer coefficients were calculated by using the double‐film theory. Decolorization kinetics were studied with model dye solutions. Decolorization experiments with a real exhausted dyebath (untreated and after biological treatment) were also carried out. The potential advantages of membrane contactors for the treatment of these types of effluents are demonstrated.
Integrating Photocatalysis and Membrane Technologies for Water Treatment
Authors: OLLIS DAVID F.
Published: July 2009

Abstract: Removal of organic contaminants in water may sometimes be more easily achieved with a pair rather than with a single unit operation. We explore here the combination of an ambient temperature, chemical oxidation photocatalysis (PC) process with physical separation via a membrane operation, microfiltration (MF), ultrafiltration (UF), or reverse osmosis (RO) using both a conceptual statement and a literature review format. Four configurations are noted and discussed here: (1) PC + MF for catalyst slurry recycle, (2) PC + UF for catalyst slurry and (polymer) reactant recycle, (3) immobilized PC and UF/RO for reactant recycle, and (4) immobilized PC on UF/RO membrane for membrane self‐cleaning. Although the literature review is encouraging with respect to plausibility, the paucity of examples indicates a need for substantial effort to fully exploit the suggested possibilities for process development.
Computer‐Aided Simulation and Design of Nanofiltration Processes
Authors: NORONHA MOHAN , MAVROV VALKO , CHMIEL HORST
Published: July 2009

Abstract: The modelling of membrane filtration processes is often performed by applying black‐box models or short‐cut methods, because of the complexity of the molecular interactions on and inside the membrane. The assumptions made for short‐cut methods can be applied with accuracy to reverse osmosis processes, whereas the simulation of nanofiltration can lead to unreliable results that sometimes deviate from real conditions to a great extent. A steady‐state process simulation, NF‐PROJECT, based on input information from membrane characterization, was developed (isothermal operation). The individual separation characteristics of each membrane element are calculated in an iterative sequence, illustrating the successive reduction in permeability and rejection between the elements arranged inside the pressure vessel. The simulation provides information on the increasing feed concentration and osmotic pressure, the hydraulic pressure loss, the deterioration of the flow conditions in the vessel, and the joint performance of the membrane elements to be analyzed. Taking an example from a practical application, a two‐stage nanofiltration pilot plant was simulated, the results of which are presented in this article. Examples of optimization potentials are illustrated for the target criteria of economic efficiency (specific energy costs), permeate quality, and flow.
Material Transport through Charged Mosaic Membrane
Authors: YAMAUCHI AKIRA , FUKUDA TAKASHI
Published: July 2009

Abstract: Several characteristics of a charged mosaic membrane with parallel array of negative and positive charges were investigated by using transport studies and the related analysis. From an analysis of the volume flux and salt flux based on irreversible thermodynamics, preferential salt transport across the charged mosaic membrane was clearly demonstrated. Additionally, transport properties of amino acids and sucrose through the charged mosaic membrane were estimated, relatively, on the basis of KCl transport. As a result, amino acid transport depends largely on the charged states and molecular weight; however, for sucrose transport, non‐electrolyte was rejected under all experimental conditions.
Stable Liquid Membranes
Authors: KOVVALI A. SARMA , SIRKAR KAMALESH K.
Published: July 2009

Abstract: Immobilized liquid membranes offer the advantages of high selectivity and species flux in separation of species from either gaseous or liquid streams. Despite these advantages, they are not yet commercially viable because of their instabilities and limited lifetime. This paper presents recent advances made in developing stable immobilized liquid membranes for gas separation with high fluxes and selectivities for the species of interest. Some of the recent approaches being studied for development of stable liquid membranes for liquid separation are also discussed.
Poly(Vinyl Alcohol)‐Based Polyelectrolyte Pervaporation Membranes
Authors: SUN BENHUI , ZOU JIAN
Published: July 2009

Abstract: By modifying poly(vinyl alcohol) (PVA), phosphatic anionic PVA (P‐PVA) and quaternary ammonium cationic PVA (C‐PVA) with various degrees of substitution (D.S.) were synthesized. The effects of synthesis conditions on the degree of substitution were studied. With these two kinds of materials, polyelectrolyte complexes were formed and their solubilities in water were studied. Pervaporation composite membranes were prepared from P‐PVA, C‐PVA, and their polyelectrolyte complexes. Some of the membranes showed good separation performance. The polyelectrolyte complex membrane prepared by mixing P‐PVA (D.S. 2.3%) and C‐PVA (D.S. 2.9%) with weight ratio of 1/1, showed a permeation rate of 378g/m2h and separation factor of 2,250 for dehydration of ethanol/water mixture (ethanol 95.4 wt%) at a feed temperature of 75°C. Some factors that influenced pervaporation performance included type of polyelectrolyte, degree of substitution, ratio of polyanion and polycation, feed temperature, feed concentration, and elapsed time. Solvent resistance of pervaporation composite membranes was also evaluated.
Enzyme Transmission during Crossflow Filtration of Yeast Suspensions Using Gas/Liquid Two‐Phase Flows
Authors: MERCIER‐BONIN MURIEL , FONADE CHRISTIAN
Published: July 2009

Abstract: The optimal conditions for recovery of an enzyme were determined using gas/liquid two‐phase flows. When filtering the enzyme‐only solution under single‐phase flow conditions, severe fouling occurred. This fouling was manifest as a decline in flux to less than 2% of the initial water flux and a decline in protein concentration in the permeate to 30% of its initial value, during a five‐hour filtration period. When yeast cells were added under the same experimental conditions, enzyme transmission was maintained at 100% for the five‐hour period and the enzyme mass flux was twofold higher. During gas‐sparged microfiltration of the enzyme/yeast mixture in a permeate‐recycling mode at the same liquid flow rate, gas/liquid slug flow strongly decreased the transmission of the enzyme (70% decrease), even though the permeate flux was improved (140% improvement). As a result, the mass flux of the enzyme was significantly reduced. However, with a bubble flow pattern, the permeate flux was 1.5 times higher and the transmission was maintained at a high level. The enzyme mass flux was then 25% higher when compared to single‐phase flow filtration conditions. During diafiltration experiments with a bubble flow pattern, a 13% higher enzyme recovery was achieved.
Creation versus Destruction of T Cell Epitopes in the Class II MHC Pathway
Authors: WATTS C. , MOSS C. X. , MAZZEO D. , WEST M. A. , MATTHEWS S. P. , LI D. N. , MANOURY B.
Published: July 2009

Abstract: Proteases perform two key roles in the class II MHC antigen processing pathway. They initiate removal of the invariant chain chaperone for class II MHC and they generate peptides from foreign and self proteins for eventual capture and display to T cells. How a balance is achieved between generation of suitable peptides versus their complete destruction in an aggressive proteolytic environment is not known. Nor is it known in most cases which proteases are actually involved in antigen processing. Our recent studies have identified asparagine endopeptidase (AEP or legumain) as an enzyme that contributes to both productive and destructive antigen processing in the class II MHC pathway. The emerging consensus seems to be that individual proteolytic enzymes make clear and non‐redundant contributions to antigen processing.
Dendritic Cell Function in Vivo during the Steady State: A Role in Peripheral Tolerance
Authors: STEINMAN RALPH M. , HAWIGER DANIEL , LIU KANG , BONIFAZ LAURA , BONNYAY DAVID , MAHNKE KARSTEN , IYODA TOMONORI , RAVETCH JEFFREY , DHODAPKAR MADHAV , INABA KAYO , NUSSENZWEIG MICHEL
Published: July 2009

Abstract: The avoidance of autoimmunity requires mechanisms to actively silence or tolerize self reactive T cells in the periphery. During infection, dendritic cells are not only capturing microbial antigens, but also are processing self antigens from dying cells as well as innocuous environmental proteins. Since the dendritic cells are maturing in response to microbial and other stimuli, peptides will be presented from both noxious and innocuous antigens. Therefore it would be valuable to have mechanisms whereby dendritic cells, prior to infection, establish tolerance to those self and environmental antigens that can be processed upon pathogen encounter. In the steady state, prior to acute infection and inflammation, dendritic cells are in an immature state and not fully differentiated to carry out their known roles as inducers of immunity. These immature cells are not inactive, however. They continuously circulate through tissues and into lymphoid organs, capturing self antigens as well as innocuous environmental proteins. Recent experiments have provided direct evidence that antigen‐loaded immature dendritic in vivo silence T cells either by deleting them or by expanding regulatory T cells. In this way, it is proposed that the immune system overcomes at least some of the risk of developing autoimmunity and chronic inflammation. It is proposed that dendritic cells play a major role in defining immunologic self, not only centrally in the thymus but also in the periphery.
Opsonization of Apoptotic Cells and Its Effect on Macrophage and T Cell Immune Responses
Authors: KIM SUN JUNG , GERSHOV DEBRA , MA XIAOJING , BROT NATHAN , ELKON KEITH B.
Published: July 2009

Abstract: Genetic studies in mice indicate that predisposition to lupus‐like diseases is caused by at least three mechanisms: (1) alterations in the threshold of activation of lymphocytes or macrophages; (2) defective signaling for activation‐induced cell death; and (3) reduced clearance of apoptotic cells. To define the mechanisms whereby lupus develops in mice with deficiencies in either C1q, serum amyloid P component (SAP, the mouse counterpart of C‐reactive protein, or CRP), or serum IgM, we studied the efficiency of phagocytosis of apoptotic cells using serum with varying levels of C1q, CRP, or IgM; we also examined the immune response to ingestion of dying cells under these conditions. Deficiency of C1q led to impaired macrophage phagocytosis of apoptotic cells, whereas CRP augmented phagocytosis, largely through recruitment of the early complement components. Like CRP, normal polyclonal IgM bound to apoptotic cells and activated complement on the cell surface. Similarly, direct binding as well as absorption experiments revealed that CRP and IgM antibodies had a similar ligand recognition specificity, namely lysophospholipids containing phosphorylcholine. IL‐12 provides a pivotal link between macrophages and the T cell response to ingested material. We observed that necrotic cells induced IL‐12 p40 expression, whereas phagocytosis of apoptotic cells profoundly reduced IL‐12 production from stimulated macrophages. Furthermore, soluble factors from macrophages that had ingested apoptotic cells suppressed interferon‐γ production by activated T cells. These findings suggest that phospholipid exposure on apoptotic cells promotes opsonization by serum proteins leading to activation of complement, macrophage ingestion, and T cell suppression. We discuss how deficient opsonization or processing of dying cells leads to autoimmunity.
Somatic Hypermutation and B Cell Receptor Selection in Normal and Transformed Human B Cells
Authors: KÜPPERS RALF
Published: July 2009

Abstract: From the beginning to the end, the life of B cells is dominated by selection of the cells for expression of an appropriate antigen receptor. However, recent studies revealed that there are several diseases in the human where B cells lost their dependence on a B cell receptor (BCR). In classic Hodgkin's lymphoma, the lymphoma cells presumably derive from ‘crippled’ germinal center (GC) B cells that acquired unfavorable somatic Ig gene mutations, which often render originally functional immunoglobulin (Ig) genes nonfunctional. A peculiar situation is observed among Epstein‐Barr virus (EBV)‐infected B cells in angioimmunoblastic lymphadenopathy with dysproteinaemia (AILD)‐type T cell lymphoma, where somatic hypermutation uncoupled from any selection for functionality of the BCR is observed in expanding clones. Clones of EBV‐harboring B cells that show ongoing hypermutation during proliferation and are Ig‐deficient in at least a fraction of cases were recently also identified in post‐transplant lymphoproliferative disorders. Hence, transformed B cells may, in particular settings, escape the normal selectional forces to express a BCR, and EBV may cause dramatic changes in B cell differentiation programs. Somatic hypermutation may be involved in lymphomagenesis by several means. Some chromosomal translocations into Ig loci likely involve DNA‐strand breaks associated with hypermutation. Moreover, by aberrant targeting of the CD95 gene, GC B cells and lymphomas developing from them may become resistant to elimination by CD95 ligand‐expressing T cells. Finally, aberrant hypermutation of multiple proto‐oncogenes appears to be a major factor in diffuse large cell lymphoma pathogenesis.
CD137‐Mediated T Cell Co‐Stimulation Terminates Existing Autoimmune Disease in SLE‐Prone NZB/NZW F1 Mice
Authors: FOELL JUERGEN , McCAUSLAND MEGAN , BURCH JENNIFER , CORRIAZZI NICHOLAS , YAN XIAO‐JIE , SUWYN CAROLYN , O'NEIL SHAWN P. , HOFFMANN MICHAEL K. , MITTLER ROBERT S.
Published: July 2009

Abstract: T cell receptor recognition of antigen and major histocompatibility complex (signal 1) and T cell co‐stimulation (signal 2) are essential for full T cell activation, differentiation, and survival of naïve and activated T cells. The proto‐typical T cell co‐stimulatory receptor, CD28, is a constitutively expressed type I integral transmembrane protein and member of the Ig superfamily. Since its discovery, additional T cell co‐stimulatory receptors have been identified, a number of which belong to the tumor necrosis factor receptor superfamily. Included within this group is CD137 (4–1BB), an activation‐inducible, type I transmembrane protein. Co‐stimulation of T cells through CD137 effectively up‐regulates CD8 T cell activation and survival. Although CD4+ T cells are efficiently activated through the T cell receptor and CD137 receptor, it provokes CD4+ T cell anergy and blockade of T‐dependent humoral immune responses. Therefore, we tested whether agonistic anti‐CD137 monoclonal antibodies (mAbs) would be effective in blocking the induction or progression of B cell dependent autoimmune disease. Herein, we demonstrate the protective effect of agonistic anti‐CD137 mAbs in blocking systemic lupus erythematosus (SLE) disease progression in NZB/W F1 mice. Protection from SLE following anti‐CD137 mAb treatment is not confined to rescuing mice from disease progression; rather, it fully protects young mice from developing any symptoms of disease. We further found that treatment of proteinuric mice with anti‐CD137 blocks ongoing anti‐dsDNA autoantibody production.
On Explanatory Practice and Disciplinary Identity
Authors: WOODY ANDREA I.
Published: July 2009

Abstract: This essay explores connections between explanatory discourse and disciplinary identity. While explanation is frequently assumed to be one of the central aims of the sciences, I contend that if we want to appreciate explanation's role within scientific practice, we need to look elsewhere. Explanations encode the aims and values of particular scientific communities, telling practitioners what they should want to know about the world and how they should reason to get there. By inculcating patterns of reasoning consistent with these aims and values, explanatory discourse is crucial for the formation of cohesive scientific disciplines. Yet because such discourse is frequently grounded in exemplars, the implicit nature of exemplar‐based training must be confronted. I argue that exemplars are an appropriate vehicle for communicating skill‐based knowledge and methodological norms and, additionally, that exemplars are a practical means of grappling with the indeterminacy of theory application.
Chemical Substances and Intensive Properties
Authors: NEEDHAM PAUL
Published: July 2009

Abstract: Despite the importance molecular structure has acquired in 20th century chemistry, more traditional macroscopic notions in terms of a continuous concept of matter continue to play a role in chemical theorizing. In the light of the extensive and determined criticism of reductionism in recent philosophy of chemistry, it is of interest to see macroscopic ontology treated autonomously. One aspect of this is developed here, namely, the concept of chemical substance. This is characterized by contrast with phases and solutions. The key conception is that of an intensive property, which is defined by appeal to the mereological structure of parts of the entities bearing substance properties.
Explanation in Organic Chemistry
Authors: GOODWIN WILLIAM
Published: July 2009

Abstract: In this paper, a model of a subclass of the explanations given in organic chemistry is developed. This model is supported by three concrete examples. The model suggests that in this discipline, laws, theories, and causal reasoning are interrelated in interesting and heretofore unexplored ways. The model also reserves a prominent place for idealizations and capacities ascribed on the basis of the structural features of molecules. The author hopes to have established that philosophical reflection on the methodology of organic chemistry can yield interesting and valuable new insights into classical issues in the philosophy of science.
Chemical Self‐Organization, Complexification, and Process Metaphysics
Authors: SALMON JAMES F.
Published: July 2009

Abstract: Recognizing the self‐activity of matter through complexification can lead to seeing evolution as a process of union. This process approach emphasizes a metaphysics of becoming rather than of being. Advances in the science of thermodynamics lead to understanding chemical self‐organization as a stage in this process of complexification.
ATP Binding Residues of Sarcoplasmic Reticulum Ca2+‐ATPase
Authors: McINTOSH D. B. , CLAUSEN J. D. , WOOLLEY D. G. , MacLENNAN D. H. , VILSEN B. , ANDERSEN J. P.
Published: July 2009

Abstract: ATP‐binding residues in the N and P domains of sarcoplasmic reticulum Ca‐ATPase have been investigated using mutagenesis in combination with a binding assay based on the photolabeling of Lys492 with [g‐32P] 2′,3′‐O‐(2,4,6 trinitrophenyl)‐8‐azido‐ATP and competition with nucleotide. In the N domain, mutations to several residues in conserved motifs, 438GEATE, 487FSRDRK, 515KGAPE, and 560RCLALA produce nucleotide‐binding defects. Key residues include Thr441, Glu442, Phe487, Arg489, Lys492, Lys515, Arg560, and Leu562. In the absence of Mg2+, Arg489, Lys492, and Arg560 are most important, whereas in its presence Thr441 and Glu442 also play a crucial role. In the P domain, Asp351 is striking for its strong electrostatic repulsion of the γ‐phosphate, especially in the presence of Mg2+. Lys352 is a key residue, and Asp627 and Lys684 must come close to the nucleotide. Thr353, Asn359, Asp601, and Asp703 interact only in the presence of Mg2+. Asn706 and Asp707 are unimportant for nucleotide binding. The results identify several ATP binding residues in the N and P domains and suggest that Mg2+ changes the nucleotide/protein interaction in both. Models of bound ATP and MgATP are presented.
Cation Stoichiometry and Cation Pathway in the Na,K‐ATPase and Nongastric H,K‐ATPase
Authors: HORISBERGER JEAN‐DANIEL , GUENNOUN SAÏDA , BURNAY MURIEL , GEERING KÄTHI
Published: July 2009

Abstract: The mechanism of cation translocation by the Na,K‐ATPase was investigated by cysteine scanning mutagenesis and measurements of accessibility through exposure to cysteine reagents. In the native protein, accessible residues were found only at the most extracellular residues of the 5th and 6th transmembrane segments (TMS) and the short loop between them. However, after modification by palytoxin a number of residues became accessible along the whole length of the 5th TMS and in the outer half of the 6th TMS, showing the contribution of each of these segments to the ‘channel’ formed by the palytoxin‐transformed Na,K‐pump. Assuming that this structure is similar in the native and the palytoxin‐transformed pump, our data allow us to determine the residues lining the cation pathway from the extracellular solution to their binding sites. A critical position in the 5th TMS contains a lysine conserved in all known nonelectrogenic H,K‐ATPases, and a serine in all known electrogenic Na,K‐ATPase sequences. Wild‐type or mutant Na,K‐or H,K‐ATPase a subunits were coinjected with the Bufoβ2 subunit in Xenopus oocytes and Rb86 uptake and electrophysiological measurements were performed. An electrogenic activity was recorded for the H,K‐ATPase mutants in which the positively charged lysine had been replaced by neutral or negatively charged residues, while nonelectrogenic transport was observed with the S782R mutant of the Na,K‐ATPase. The presence or the absence of a positively charged residue at the S782 position appears to be critical for the stoichiometry of cation exchange.
Toward an Understanding of Ion Transport through the Na,K‐ATPase
Authors: APELL HANS‐JÜRGEN
Published: July 2009

Abstract: In the Na,K‐ATPase the charge‐translocating reaction steps were found to be binding of the third Na+ ion to the cytoplasmic side and the release of all three Na+ ions to the extracellular side as well as binding of the two K+ ions on the extracellular side. The conformation transition E1→ E2 was only of minor electrogenicity; all other reaction steps produced no significant charge movements. In the SR Ca‐ATPase and the gastric H,K‐ATPase, all ion‐binding and ‐release steps were identified to move charge through the membrane. The high‐resolution structure of the SR Ca‐ATPase in state E1 revealed the position of the ion‐binding sites in the transmembrane part of the protein. If the same arrangement is assumed for the Na pump, the missing expected charge movements in state E1 may to be assumed to be apparent effects. With the proposal that binding of 2 Na+ or 2 K+ is compensated correspondingly by H+ ions, agreement between structural and functional aspects is obtained. Investigations of the pH‐dependence of ion‐binding steps indicate competition between the ions and electrogenic H+ binding in support of this concept.
Na,K‐Pump Reaction Kinetics at the Tip of a Patch Electrode
Authors: PELUFFO R. DANIEL , BERLIN JOSHUA R.
Published: July 2009

Abstract: Patch‐clamp electrophysiological techniques allow manipulations of electrochemical driving forces for ion transport by the Na,K‐ATPase. For this reason, this technique has been used to study steady‐state ion transport properties of the Na,K‐ATPase. High temporal resolution during these manipulations also permits rapid reactions, such as extracellular ion‐binding reactions, to be measured as charge movements when the enzyme is engaged in electroneutral ion exchange modes. Just as useful, but less widely recognized, is the ease with which electrophysiological techniques can be used to critically study reaction steps that do not directly involve ion binding. Three studies are briefly presented to show how pre‐steady‐state and/or steady‐state electrophysiological techniques can be used to study ion‐binding reactions in a novel fashion and the kinetics of electrically silent reaction steps of this enzyme. The reaction kinetics derived from each of these studies can be used to attain detailed mechanistic information about ion transport by the Na,K‐ATPase.
Adaptation of Murine Inner Medullary Collecting Duct (IMCD3) Cell Cultures to Hypertonicity
Authors: CAPASSO JUAN M. , RIVARD CHRISTOPHER J. , ENOMOTO LAURA M. , BERL TOMAS
Published: July 2009

Abstract: Recently, we have adapted IMCD3 cell cultures to survive under increasing hypertonic conditions (i.e., 600 and 900 mOsmol/kg H2O). In adapted cells, ATPase activity is increased by one order of magnitude, while the expression of the α and β subunit is increased by a factor of 4 to 5 over controls (300 mOsmol/kg H2O). Corresponding increases in mRNAs were also detected. The γ subunit has been described as being uniquely expressed in some areas of the kidney, but never in cell cultures (even those derived from kidney tissues). However, the γ subunit was detected at the protein and mRNA levels in the adapted IMCD3 cells. In contrast to the α and β subunits, the levels of γ protein and mRNA expression continue to increase as a function of the media ion concentration. We have also demonstrated that signaling pathways that upregulate the α, β, and γ subunits are very different. Increasing concentrations of the PI3 kinase inhibitor, LY294002, resulted in a dose‐dependent reduction in the expression of the γ subunit, with total abolition at 10 μM. However, LY294002 had no significant effect on the expression of the α subunit. Inhibition of the JNK2 (but not of the JNK1) pathways by dominant negative transfections abolished the upregulation of the γ, but not the a subunit. Failure to upregulate the expression of the γ subunit was associated with a marked decrease in cell viability upon stress.
Gamma Structural Variants Differentially Regulate Na,K‐ATPase Properties
Authors: ARYSTARKHOVA ELENA , WETZEL RANDALL K.
Published: July 2009

Abstract: Renal Na,K‐ATPase is tightly bound to a small regulatory protein, the gamma subunit (FXYD2). In rat, it occurs in two splice variants with different N‐termini. Immunolocalization on kidney sections revealed distinct distribution of the γ splice variants along the rat nephron. Where coexpressed, they coimmunoprecipitated with each other along with the alpha subunit, suggesting assembly in oligomeric complexes. Functional consequences of association with gamma were monitored in stably transfected NRK‐52E cells. The outcome was that splice variants can differentially modulate the major intrinsic properties of the Na,K‐ATPase under normal and stress‐related conditions. The data imply an adaptive physiological mechanism of regulation of renal Na,K‐ATPase through modulation of pump properties, gene expression, or both.
Phosphorylation of the Na+,K+‐ATPase in Skeletal Muscle
Authors: AL‐KHALILI LUBNA , KROOK ANNA , CHIBALIN ALEXANDER V.
Published: July 2009

Abstract: In skeletal muscle, insulin stimulation leads to phosphorylation of Na+,K+‐ATPase α‐subunits on both serine/threonine and tyrosine residues, translocation of Na+,K+‐ATPase molecules to the plasma membrane, and increased Na+,K+‐ATPase activity. The molecular nature of the tyrosine kinase that phosphorylates Na+,K+‐ATPase is not yet identified. In vitro phosphorylation experiments show that the α‐subunit of Na+,K+‐ATPase from skeletal muscle is a substrate for the tyrosine‐specific protein kinase c‐src. Tyrosine phosphorylation of the α‐subunits of Na+,K+‐ATPase may be an important mechanism for insulin‐mediated regulation of Na+,K+‐ATPase translocation and activity.
The Thermogenic Function of the Sarcoplasmic Reticulum Ca2+‐ATPase of Normal and Hyperthyroid Rabbit
Authors: DE MEIS LEOPOLDO , ARRUDA ANA PAULA , DA‐SILVA WAGNER S. , REIS MARCELO , CARVALHO DENISE P.
Published: July 2009

Abstract: After formation of a Ca2+ gradient, the amount of heat released during the hydrolysis of each mol of ATP cleaved (ΔHcal) varies depending on the Ca2+‐ATPase isoform expressed by the muscle cell. In vesicles derived from the sarcoplasmic reticulum of white muscle (SERCA 1) most of the ATP cleaved is not coupled to Ca2+ transport, and the ΔHcal varies between −20 and −22 kcal/mol. In contrast, in vesicles derived from red muscle (SERCA 2a) the hydrolysis of ATP is coupled with Ca2+ transport, and the ΔHcal varies between −12 and −14 kcal/mol. Hyperthyroidism increases the rate of heat production by the Ca2+‐ATPase fourfold in white muscle and 40‐fold in red muscle. In hyperthyroid rabbits, the amount of sarcoplasmic reticulum protein recovered from white and red muscle is four‐ to fivefold greater than that obtained from control rabbits. Hyperthyroid red muscle expresses SERCA 1, and the vesicles derived from these muscle hydrolyze ATP through a catalytic route that is not coupled to Ca2+ transport, thus increasing the amount of heat released during ATP hydrolysis, the ΔHcal varying between −20 and −22 kcal/mol.
Regulation of Ca2+ Signaling by Na+ Pump Alpha‐2 Subunit Expression
Authors: GOLOVINA VERA , SONG HONG , JAMES PAUL , LINGREL JERRY , BLAUSTEIN MORDECAI
Published: July 2009

Abstract: The role of the Na+ pump α2 subunit in Ca2+ signaling was examined in primary cultured astrocytes from wild type (WT) mouse fetuses and those with a null mutation in one (Het) or both (KO) α2 genes. Cytosol Na+ and Ca2+ concentrations ([Na+]CYT and [Ca2+]CYT) were measured with benzofuran isophthalate (SBFI) and Fura‐2. Het astrocytes express ∼50% of normal α2; KO cells express none. Resting [Na+]CYT= 6.5 mM in WT, 6.8 mM in Het, and 8.0 mM in KO cells; 500 nM ouabain (which inhibits only α2) equalized [Na+]CYT at 8 mM in all genotypes. Resting [Ca2+]CYT= 132 nM in WT, 162 nM in Het and 196 nM in KO cells. Cyclopiazonic acid (CPA), which inhibits endoplasmic reticulum (ER) Ca2+ pumps, induces elevation of [Ca2+]CYT. These Ca2+ responses are augmented in Het and KO cells. This correlates with α2 Na+ pump and Na+/Ca2+ exchanger (NCX) localization in plasma membrane (PM) microdomains that overlie the ER. Selective reduction of α2 Na+ pump activity apparently elevates local [Na+] and, via NCX, [Ca2+] in the tiny cytosolic space between the PM and ER. This augments adjacent ER Ca2+ stores and amplifies Ca2+ signaling without elevating bulk [Na+]CYT.
Pathogenic Mechanisms of Diseases Caused byRickettsia
Authors: WALKER DAVID H. , VALBUENA GUSTAVO A. , OLANO JUAN P.
Published: July 2009

Abstract:The specter of bioterrorism employing genetically engineeredRickettsiaresistant to all antibiotics should reawaken the world's desire to elucidate the pathogenesis of typhus and spotted fever rickettsioses in a search for mechanisms vulnerable to interdiction. The pathogenetic sequence includes rickettsial entry into the dermis, hematogenous dissemination to vascular endothelial cells (most critically in brain and lungs), increased vascular permeability, edema, and immunity mediated by NK cells, IFN‐γ, TNF‐α, RANTES, antibodies, and cytotoxic T lymphocytes. Silverman has demonstrated the role of reactive oxygen species (ROS) produced byR. rickettsii‐infected endothelial cells in peroxidative damage to cell membranesin vitro, and Heinzen has described actin‐based rickettsial intracellular mobility and intercellular spread. At this point the availability of sequences of rickettsial genomes and excellent animal models of rickettsioses have yielded insufficient progress towards the identification of rickettsial virulence factors and knowledge of the importance of injury mediated by ROS, phospholipase A2, protease(s) or other mechanismsin vivo. Attention to the rickettsiosis‐associated procoagulant state led to determination that hemostatic mechanisms largely prevent major hemorrhage without disseminated intravascular coagulation or thrombosis‐mediated ischemia. Particularly lacking is knowledge of early eventsin vivoat the portal of entry in skin (or lung), of the effects of the inoculum medium (arthropod saliva or feces), mediators produced by infected endothelium under conditions of flow and of the contributionsin vivoof immune effectors to pathology, of the role of apoptosis in rickettsial infection, and of the endothelial cell alterations that account for increased vascular permeability. The host cell receptor for theRickettsialigand and the mechanism of rickettsial escape from the phagosome need to be elucidated.
Ecology and Epidemiology of Spotted Fever Group Rickettsiae and New Data from Their Study in Russia and Kazakhstan
Authors: RUDAKOV N. V. , SHPYNOV S. N. , SAMOILENKO I. E. , TANKIBAEV M. A.
Published: July 2009

Abstract: Rickettsiae represent a wide range of pathogenicity from classic and new pathogens to endosymbionts of eukaryotic cells. Recent studies of rickettsiae have widened the number of representatives of genus Rickettsia, especially in the spotted fever group (SFG). Rickettsiae of SFG are tick‐borne microorganisms with effective transovarial and transstadial transmission. The main hosts are ticks (Dermacentor, Rhipicephalus, Haemophysalis, Ixodes, and Amlyomma). Strategy of maintenance of tick microorganisms is vector‐type transfer and tropism to endothelial cells or blood cells of animals. The main epidemiological characteristics of SFG rickettsioses are different kinds of anthropogenic activity and connection of morbidity with seasonal tick activity. Two other important characteristics are quantitative and qualitative heterogeneity of its populations (different genotypes of Rickettsia in the same territory and species of ticks, for example) and coexistence of different tick microorganisms (rickettsiae, borreliae, ehrlichiae, tick‐borne encephalitis complex viruses, etc.). The role of new rickettsial genotypes in infectious diseases is poorly udnerstood. Simultaneous study of ticks after bites, blood and skin biopsies of patients may be used for detection of spectrum of tick‐borne pathogens in mixed natural foci. Interference between rickettsiae with different virulence may affect its populations and levels of morbidity.
Scrub Typhus
Authors: LERDTHUSNEE K. , KHUNTIRAT B. , LEEPITAKRAT W. , TANSKUL P. , MONKANNA T. , KHLAIMANEE N. , INLAO I. , KENGLUECHA A. , MUNGVIRIYA S. , CHANDRANOI K. , KRAIROJANANAN P. , BODHIDATTA D. , RODKWAMTHOOK W. , PHULSUKSOMBATI D. , SANGJUN N. , WATCHARAPICHAT P. , JONES J. W. , COLEMAN R. E.
Published: July 2009

Abstract: A total of 4,281 rodents, belonging to 20 species, was collected from three villages in Chiangrai Province, northern Thailand, from January 1994 to December 2001. The predominant species were Rattus rattus, R. losea and Bandicota indica, accounting for 96% of the total. More than 135,000 chigger mites were removed from wild rodents of which 1% were identified as Leptotrombidium chiangraiensis, a new species vector of scrub typhus. Five Orientia tsutsugamushi‐infected L. chiangraiensis colonies established from chiggers removed from R. rattus and R. losea rodents. The prevalence of O. tsutsugamushi infection in colonies ranged from 7–89%. Vertical and horizontal transmission efficacies of O. tsutsugamushi were stable between generations among colonies of L. chiangraiensis. The two isolates of O. tsutsugamushi obtained from individual adult L. chiangraiensis mites represent the first successful isolation of this bacterium from individual adult mites. Characterization of the O. tsutsugamushi isolates is under way.
Rickettsialpox in New York City
Authors: PADDOCK CHRISTOPHER D. , ZAKI SHERIF R. , KOSS TAMARA , SINGLETON JOSEPH , SUMNER JOHN W. , COMER JAMES A. , EREMEEVA MARINA E. , DASCH GREGORY A. , CHERRY BRYAN , CHILDS JAMES E.
Published: July 2009

Abstract: Rickettsialpox, a spotted fever rickettsiosis, was first identified in New York City (NYC) in 1946. During the next five years, approximately 540 additional cases were identified in NYC. However, during the subsequent five decades, rickettsialpox received relatively little attention from clinicians and public health professionals, and reporting of the disease diminished markedly. During February 2001 through August 2002, 34 cases of rickettsialpox in NYC were confirmed at CDC from cutaneous biopsy specimens tested by using immunohistochemical (IHC) staining, PCR analysis, and isolation of Rickettsia akari in cell culture, as well as an indirect immunofluorescence assay of serum specimens. Samples were collected from patients with febrile illnesses accompanied by an eschar, a papulovesicular rash, or both. Patients originated predominantly from two boroughs (Manhattan and the Bronx). Only 8 (24%) of the cases were identified prior to the reports of bioterrorism‐associated anthrax in the United States during October 2001, and lesions of several patients evaluated during and subsequent to this episode were suspected initially to be cutaneous anthrax. IHC staining of biopsy specimens of eschars and papular lesions were positive for spotted fever group rickettsiae for 32 patients. Of the eleven patients for whom paired serum samples were obtained, all demonstrated fourfold or greater increases in antibody titers reactive with R. akari. The 17‐kDa protein gene sequence of R. akari was amplified from eschars of five patients. Four isolates of R. akari were obtained from cutaneous lesions. Possible factors responsible for the increase in clinical samples evaluated for rickettsialpox during this interval include renewed clinical interest in the disease, improved diagnostic methods, epizootiological influences, and factors associated with the recent specter of bioterrorism.
Isolation of a Rickettsia Related to Astrakhan Fever Rickettsia from a Patient in Chad
Authors: FOURNIER PIERRE‐EDOUARD , XERIDAT BERNARD , RAOULT DIDIER
Published: July 2009

Abstract: We isolated a novel spotted fever group rickettsia from a patient coming back from Chad with fever and a maculopapulous rash. In Africa, only six pathogenic spotted fever group rickettsiae have been identified, R. conorii, R. africae, R. akari, R. aeschlimannii, ‘R. mongolotimonae,’ and R. felis. Our isolate was identified by PCR amplification and sequencing of the 16S rRNA (16S rDNA), citrate synthase (gltA), and rOmpA (ompA) encoding genes. The 16S rDNA, gltA, and ompA sequences of the isolate were found to be 99.7, 99.6, and 99.5% identical with that of Astrakhan fever rickettsia, respectively. This rickettsia is endemic in the Caspian sea area and has also recently been identified in Kosovo. Using mouse serotyping, the currently accepted method for the identification of spotted fever group rickettsiae, the Chad isolate exhibited a specificity difference of 2 when compared to Astrakhan fever rickettsia and at least 4 when compared with other members of the R. conorii complex. The Chad isolate should be considered a variant of Astrakhan fever rickettsia. This is the first description of Astrakhan fever rickettsia outside Europe and the bacterium may be responsible for cases of spotted fever in Chad. Although Astrakhan fever rickettsia is transmitted by Rhipicephalus ticks in Europe, further studies are indicated to identify its vector in Africa where these ticks are also prevalent.
Clinical Impact of PersistentBartonellaBacteremia in Humans and Animals
Authors: CHOMEL BRUNO B. , KASTEN RICKIE W. , SYKES JANE E. , BOULOUIS HENRI‐JEAN , BREITSCHWERDT EDWARD B.
Published: July 2009

Abstract:Bartonellaspp. are emerging vector‐borne pathogens that cause persistent, often asymptomatic bacteremia in their natural hosts. As our knowledge progresses, it appears that chronic infection may actually predispose the host to mild, insidious nonspecific manifestations or induce, in selected instances, severe diseases. Persistent asymptomatic bacteremia is most common in animals that serve as the main reservoir for the specificBartonella. In humans, these organisms areB. bacilliformisandB. quintana. OtherBartonellaspecies, for which humans are not the natural reservoir, tend to cause persistent bacteremia only in immunodeficient individuals. In some of these individuals, endothelial cell proliferation may create lesions such as bacillary angiomatosis or bacillary peliosis. In cats, bacteremia of variable level and continuity may last for years. Some strains ofB. henselaemay induce clinical manifestations, including fever, mild neurological signs, reproductive disorders, whereas others do not induce clinically obvious disease. Reproductive disorders have also been reported in mice experimentally infected withB. birtlesii. Finally, canids constitute the most interesting naturally occurring animal model for the human disease. Like immunocompetent people, healthy dogs only occasionally demonstrate long‐term bacteremia when infected withBartonellaspp. However, some dogs develop severe clinical manifestations, such as endocarditis, and the pathologic spectrum associated withBartonellaspp. infection in domestic dogs is rapidly expanding and resembles the infrequently reported clinical entities observed in humans. In coyotes, persistent bacteremia is more common than in domestic dogs. It will be of interest to determine if coyotes develop clinical or pathological indications of infection.
Physician Knowledge of the Diagnosis and Management of Rocky Mountain Spotted Fever
Authors: O'REILLY M. , PADDOCK C. , ELCHOS B. , GODDARD J. , CHILDS J. , CURRIE M.
Published: July 2009

Abstract: Rocky Mountain spotted fever (RMSF), a tick‐borne illness that has its highest incidence in the south central and southeastern United States, is often a diagnostic challenge, as patients frequently present with nonspecific symptoms during the early stages of illness. RMSF has a high case fatality rate among untreated individuals, and the median time from onset of symptoms to death is only eight days, making early recognition and treatment of RMSF crucial. In two Mississippi public health districts, 148 primary care physicians were randomly selected and mailed surveys regarding RMSF diagnosis, treatment, and prevention. Eighty‐four of the 148 (57%) physicians responded. Responses from different specialties and different health districts were compared using chi square statistics. Almost all (99%) physicians correctly identified doxycycline as the antibiotic agent of choice for treating adults and adolescents. However, only 21% of family practice physicians, and 25% of emergency medicine physicians correctly identified the antibiotic of choice for treating children with RMSF. Twenty‐three percent of physicians responded that waiting for the development of a rash before prescribing antibiotics is an appropriate treatment strategy. The current standard of care—doxycycline as the agent of choice among children 8 years of age or younger with suspected RMSF—has not been effectively communicated to all physicians caring for children. Also, many physicians are not familiar with the rationale underlying initiation of antibiotic therapy prior to the development of rash in patients with suspected RMSF. Continuing education efforts should focus on antibiotic selection in pediatric patients and initiation of therapy prior to the onset of rash in appropriate patients.