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The crystal structure of the dimeric membrane domain of human Band 3 1 , the red cell chloride/bicarbonate anion exchanger 1 (AE1, SLC4A1), provides a structural context for over four decades of studies into this historic and important membrane glycoprotein. In this review, we highlight the key structural features responsible for anion binding and translocation and have integrated the following topological markers within the Band 3 structure: blood group antigens, N-glycosylation site, protease cleavage sites, inhibitor and chemical labeling sites, and the results of scanning cysteine and N-glycosylation mutagenesis. Locations of mutations linked to human disease, including those responsible for Southeast Asian ovalocytosis, hereditary stomatocytosis, hereditary spherocytosis, and distal renal tubular acidosis, provide molecular insights into their effect on Band 3 folding. Finally, molecular dynamics simulations of phosphatidylcholine self-assembled around Band 3 provide a view of this membrane protein within a lipid bilayer. [ABSTRACT FROM AUTHOR]

Mucosal immunity has a crucial role in controlling human respiratory tract infections. This study characterizes the naturally acquired mucosal antibody levels to three Streptococcus pneumoniae (Spn) protein antigens, pneumococcal histidine triad protein D (PhtD), pneumococcal choline binding protein A (PcpA), and pneumolysin (Ply), and assesses the association of the mucosal antibody levels with occurrence of acute otitis media (AOM) caused by Spn. Both nasopharyngeal (NP) immunoglobulin G (IgG) and IgA levels to all three proteins slightly decreased in children from 6 to 9 months of age and then gradually increased through 24 months of age. Spn NP colonization was associated with higher mucosal antibody levels to all three proteins. However, children with Spn AOM had 5-8-fold lower IgG and 3-6-fold lower IgA levels to the three proteins than children without AOM but asymptomatically colonized with Spn. Antigen-specific antibody levels in the middle ear fluid (MEF) were correlated with antibody levels in the NP. Children with AOM caused by Spn had lower antibody levels in both the MEF and NP than children with AOM caused by other pathogens. These results indicate that higher naturally acquired mucosal antibody levels to PhtD, PcpA and Ply are associated with reduced AOM caused by Spn. [ABSTRACT FROM AUTHOR]

Abstract: Picophytoplankton carbon biomass at the Bermuda Atlantic Time-series Study (BATS) site from June 2004 to December 2010 was estimated from the direct calibration of cellular carbon content and forward light scatter (via flow cytometry). Seasonality and interannual dynamics of Prochlorococcus, Synechococcus and small eukaryotic algae (<12μm diameter) abundance, cellular carbon content (Q C ; particulate organic carbon; POC cell−1), and group-specific carbon biomass are reported. Q C of individual taxa varied with depth and season by as much as an order of magnitude, roughly comparable to variability in abundance. During the time-series there were obvious shifts in the taxonomic distribution of photosynthetic carbon biomass; these interannual shifts in biomass were due to simultaneous changes in both Q C and cell abundance. The observed pattern was not apparent from numerical abundance alone, highlighting the importance of Q C measurements in place of using fixed conversion factors to better understand biological carbon dynamics. Changes in the phase of the North Atlantic Oscillation (NAO) from positive to negative modes correlated with shifts in biomass between picocyanobacteria and small eukaryotic algae, respectively. Thus, shifts in algal community structure are inferred to be associated with changes in light intensity and implied nutrient supply via mixing (i.e., patterns in upper ocean stability). These observed changes in phytoplankton biomass partitioning were correlated with the important ocean carbon cycle parameters of export flux, mesopelagic transfer efficiency, and elemental stoichiometry. Importantly, interannual relationships between these parameters and algal biomass were detected only when Q C was considered as variable. [Copyright &y& Elsevier]

Abstract: Brookhaven National Laboratory is in the process of building a new Electron storage ring for scientific research using synchrotron radiation. This facility, called the “National Synchrotron Light Source II” (NSLS-II), will provide x-ray radiation of ultra-high brightness and exceptional spatial and energy resolution. It will also provide advanced insertion devices, optics, detectors, and robotics, designed to maximize the scientific output of the facility. The project scope includes the design of an electron storage ring and the experimental beamlines, which stores a maximum of 500mA electron beam current at an energy of 3.0GeV. When fully built there will be at least 58 beamlines using synchrotron radiation for experimental programs. It is planned to operate the facility primarily in a top-off mode, thereby maintaining the maximum variation in the synchrotron radiation flux to <1%. Because of the very demanding requirements for synchrotron radiation brilliance for the experiments, each of the 58 beamlines will be unique in terms of the source properties and experimental configuration. This makes the shielding configuration of each of the beamlines unique. The shielding calculation methodology and the results for five representative beamlines of NSLS-II, have been presented in this paper. [Copyright &y& Elsevier]

Abstract: Dynamic behavior in mobile telecommunications markets is often the result of interactions between market actors. As the structure and dynamic complexity of such interactions tend to be difficult to identify, it is challenging for regulators to anticipate the effects of their policy decisions. This article uses system dynamics to evaluate the effect of technology harmonization and mobile number portability policies on mobile voice diffusion and service competition. This is realized by first modeling the endogenous feedback structure resulting in dynamic behavior in the mobile telecommunications market and then by configuring the model for retrospective simulations of the Finnish market. Based on the retrospective simulations, the effect of technology harmonization and mobile number portability policy decisions is analyzed and the usefulness of system dynamics is demonstrated. [Copyright &y& Elsevier]

Abstract: Brookhaven National Laboratory is designing a new electron synchrotron for scientific research using synchrotron radiation. This facility, called the “National Synchrotron Light Source II” (NSLS-II), will provide x-ray radiation of ultra-high brightness and exceptional spatial and energy resolution. It will also provide advanced insertion devices, optics, detectors and robotics, and a suite of scientific instruments designed to maximize the scientific output of the facility. The project scope includes the design, construction, installation, and commissioning of the following accelerators: a 200MeV linac, a booster synchrotron operating from 200MeV to 3.0GeV, and the storage ring which stores a maximum of 500mA current of electrons at an energy of 3.0GeV. It is planned to operate the facility primarily in a top-off mode, thereby maintaining the maximum variation in stored beam current to <1%. Because of the very demanding requirements for beam emittance and synchrotron radiation brilliance, the beam life-time is expected to be quite low, on the order of 2h. Analysis of the bulk shielding for operating this facility and the input parameters used for this analysis have been discussed in this paper. The characteristics of each of the accelerators and their operating modes have been summarized with the input assumptions for the bulk shielding analysis. [Copyright &y& Elsevier]

Abstract: Non-typeable Haemophilus influenzae (NTHi) is the most common bacteria responsible for episodic acute otitis media (AOM; non-otitis prone), recurrent AOM (rAOM; otitis prone) and AOM treatment failure (AOMTF) in children. In this 3.5 years of prospective study, we measured the serum antibody response to outer membrane proteins D, P6 and OMP26 of NTHi in children with AOM (n =26), rAOM (n =32), AOMTF (n =27). The geometric mean titers (GMTs) of IgG at their acute AOM visit against Protein D in otitis prone children were significantly lower compared to AOMTF (p value<0.01) and non-otitis prone (p value<0.03) children; otitis prone children had significantly lower IgG levels to P6 compared to AOMTF children (p value < 0.02); otitis prone children had significantly lower IgG levels to OMP26 compared to AOMTF children (p value<0.04). Comparing acute to convalescent titers after AOM, otitis prone and AOMTF children had no significant change in total IgG against all the three proteins, while non-otitis prone children had significant increases to Protein D. Anti-protein D, P6 and OMP26 antibody levels measured longitudinally during NP colonization between age 6 and 24 months in 10 otitis prone children and 150 non-otitis prone children showed <2-fold increases over time in otitis prone children compared to >4 fold increases in the non-otitis prone children (p value<0.001). We conclude that otitis prone children mount less of an IgG serum antibody response toward Protein D, P6 and OMP26 after AOM which may account for recurrent infections. The data on acute sera of otitis prone vs non-otitis prone children and the acute-to-convalescence response in non-otitis prone children point to a possible link of anti-PD to protection. Moreover, the data suggest that otitis prone children should be evaluated for their responses to Protein D, P6 and OMP26 vaccine antigens of NTHi. [ABSTRACT FROM AUTHOR]

Band 3 (Anion Exchanger 1, AE1), the predominant protein of erythrocyte membranes, facilitates Cl−/HCO 3 − exchange and anchors the plasma membrane to the cytoskeleton. The Band 3 crystal structure revealed the amino acid 812–830 region as intracellular, conflicting with protein chemical data that suggested extracellular disposition. Further, circulating senescent cell auto-antibody that cannot enter erythrocytes, binds two regions of Band 3: residues 538–554 and 812–830. To reconcile this discrepancy, we assessed localization of residues 812–830 with Band 3 expressed in HEK293 cells and human erythrocytes, using chemical labeling probes and an antibody against residues 812–830. Antibody and chemical probes revealed reorientation of 812–830 region between extracellular and intracellular. This dramatic conformational change is an intrinsic property of the Band 3 molecule, occurring when expressed in HEK293 cells and without the damage that occurs during erythrocyte circulation. Conditions used to crystallize Band 3 for structural determination did not alter conformational dynamics. Collectively, these data reveal large Band 3 conformational dynamics localized to a region previously identified as an erythrocyte senescence epitope. Surface exposure of the senescence epitope (812–830), limited by conformational dynamics, may act as the "molecular clock" in erythrocyte senescence. [Display omitted] • One region of erythrocyte membrane Band 3 protein has controversial topology. • This region binds circulating antibodies to signal cell senescence. • We tested the region's location, using confocal microscopy, and protein chemistry. • The region adopts dramatically different conformations: cytosolic and extracellular. • This conformational change may act as a molecular clock for cell senescence. [ABSTRACT FROM AUTHOR]

Summary: Background: A framework for evaluating the efficacy of antibiotics in development as well as those currently approved for acute otitis media (AOM) is needed. Objective: Review strengths and limitations of various antibiotic trial designs and their outcome measures. Methods: A review of 157 published trials involving 36,710 subjects for the treatment of AOM. Results: AOM trials have three designs: (1) clinical, clinical diagnosis and assessment of outcomes; (2) single tympanocentesis, microbiologic diagnosis (by middle ear fluid culture) and clinical assessment of outcomes; and (3) double tympanocentesis, microbiologic diagnosis and microbiologic outcome assessment. Identifiable strengths and limitations of each design are reviewed. Case definitions for entry of children in trials of AOM vary widely. The lack of stringent diagnostic criteria in a clinical design allows for inclusion of a significant proportion of children with a non-bacterial etiology (i.e., viral AOM or otitis media with effusion). Tympanocentesis increases diagnostic accuracy at study entry; however, the procedure is confounding because of its potentially therapeutic benefit and the procedure is not performed in a uniform manner. A second tympanocentesis allows a high sensitivity to detect microbiologic eradication, but it does not correlate with clinical outcomes in half of the cases. The timing of outcome assessment also varies widely among trials. Conclusions: Improved clinical diagnosis criteria for AOM are needed to enhance specificity; emphasis on a bulging tympanic membrane has the best evidence base. Tympanocentesis within study designs has merits. At study entry it assures diagnostic accuracy but may alter outcomes and it is useful to document microbiologic outcomes but lacks specificity for clinical outcomes. For all designs, test of cure assessment 2–7 days after completion of therapy seems most appropriate. [Copyright &y& Elsevier]

Abstract: In a meta-analysis of 5 randomized controlled trials involving 1030 adults, the likelihood of bacteriologic eradication in the treatment of group A β-hemolytic streptococcal (GAS) tonsillopharyngitis with 5 days of select cephalosporins (cefpodoxime, cefuroxime, cefotiam, and cefdinir) was noninferior to 10 days of penicillin (odds ratio, 1.46; 95% confidence interval, 0.96–2.22, P = 0.08). [Copyright &y& Elsevier]

Background & Aims: The revised Bethesda guidelines for Lynch syndrome recommend microsatellite instability (MSI) testing all colorectal cancers in patients diagnosed before age 50 years and colorectal cancers diagnosed in patients between ages 50 and 59 years with particular pathology features. Our aim was to identify pathology and other features that independently predict high MSI (MSI-H). Methods: Archival tissue from 1098 population-based colorectal cancers diagnosed before age 60 years was tested for MSI. Pathology features, site, and age at diagnosis were obtained. Multiple logistic regression was performed to determine the predictive value of each feature, as measured by an odds ratio (OR), from which a scoring system (MsPath) was developed to estimate the probability a colorectal cancer is MSI-H. Results: Fifteen percent of tumors (162) were MSI-H. Independent predictors were tumor-infiltrating lymphocytes (OR, 9.1; 95% confidence interval [CI], 5.9–14.1), proximal subsite (OR, 4.7; 95% CI, 3.1–7.3), mucinous histology (OR, 2.8; 95% CI, 1.7–4.8), poor differentiation (OR, 1.9; 95% CI, 1.2–3.1), Crohn’s-like reaction (OR, 1.9; 95% CI, 1.2–2.9), and diagnosis before age 50 years (OR, 1.9; 95% CI, 1.3–2.9). MsPath score ≥1.0 had a sensitivity of 93% and a specificity of 55% for MSI-H. Conclusions: The probability an individual colorectal cancer is MSI-H is predicted well by the MsPath score. There is little value in testing for DNA mismatch repair loss in tumors, or for germline mismatch repair mutations, for colorectal cancers diagnosed in patients before age 60 years with an MSPath score <1 (approximately 50%). Pathology can identify almost all MSI-H colorectal cancers diagnosed before age 60 years. [Copyright &y& Elsevier]

AE1 facilitates Cl-/HCO3- exchange across the erythrocyte membrane. To identify residues involved in substrate selection and translocation, we prepared an array of single cysteine mutants in an otherwise cysteineless background. These mutants spanning the C-terminal portion of the AE1 membrane domain from Phe806Cys885 were characterized for functional activity when expressed in human embryonic kidney 293 cells by measurement of changes of intracellular pH associated with bicarbonate transport. To identify residues involved in substrate translocation, transport activity was assessed for each mutant before and after treatment with the following sulfhydryl reagents: anionic parachloromercuibenzenesulfonate; permeant (2-aminoethyl)methanethiosulfonate; and cationic [2-(trimethylammonium)ethyl]methanethiosulfonate (MTSET). Among the 80 mutants, only certain key residues in the Val849Leu863 region were inhibited by the sulfhydryl reagent, consistent with direct involvement of these sites in anion transport. In the last two transmembrane segments, only mutants in the extracellular portion of the transmembrane segments could be inhibited by sulfhydryl reagent, suggesting that the outer portions line the translocation channel and the inner portions have some other role. Sensitivity to cationic MTSET and effects of Cl- identified the substrate charge filter as Ser852-Leu857. [ABSTRACT FROM AUTHOR]

Abstract: Current treatment guidelines from the Infectious Diseases Society of America, the American Heart Association, and the American Academy of Pediatrics recommend only oral penicillin V or intramuscular benzathine penicillin G as the drugs of choice for treatment of group A β-hemolytic streptococcal (GABHS) pharyngitis. Ten-day treatment courses with 1st-generation oral cephalosporins or erythromycin are recommended as suitable alternatives in patients who are allergic to penicillin. Despite these recommendations, oral cephalosporins are used as drugs of choice for many patients with GABHS pharyngitis. Simpler and/or short-course regimens of cephalosporins that have been approved by the Food and Drug Administration offer alternatives with the potential for unchanged patient compliance. Increasing cephalosporin use in patients with GABHS pharyngitis has followed from numerous reports and metaanalyses of cephalosporin superiority over penicillin for bacteriologic eradication and clinical response. This review examines the evidence supporting the use of cephalosporins as a first choice of treatment for many patients with GABHS pharyngitis. [Copyright &y& Elsevier]

Sexual experience in male rats followed by a period of abstinence causes sensitization to d -Amphetamine (Amph) reward, evidenced by an increased conditioned place preference (CPP) for low doses of Amph. Moreover, sexual experience induces neural plasticity within the nucleus accumbens (NAc), including induction of deltaFosB, which plays a key role in Amph reward cross-sensitization. The NMDA receptor subunit NR1 is also upregulated by mating, but the functional relevance of NMDA receptors in sex experience-induced effects is unknown. Here, we examined the influence of intra-NAc MK 801 infusions on sex experience-induced NAc deltaFosB and cFos expression, as well as mating- and Amph-induced CPP in adult male rats. In experiment 1, males received MK 801 or saline into the NAc during each of 4 consecutive days of mating or handling and were tested for Amph CPP and experience-induced deltaFosB 10 days later. Intra-NAc MK 801 during sexual behavior prevented experience-induced increases in Amph CPP and NAc deltaFosB expression without affecting sexual behavior. In experiment 2, the effects of intra-NAc MK 801 on mating-induced CPP were examined by intra-NAc infusion of MK 801 or saline prior to mating on conditioning days. Intra-NAc MK 801 did not affect mating-induced CPP. Next, effects of intra-NAc MK 801 on mating-induced cFos immunoreactivity were examined. MK 801 prevented mating-induced cFos expression in NAc shell and core. Together, these results provide evidence that NAc NMDA receptor activation during sexual behavior plays a key role in mating-induced cFos and deltaFosB expression and subsequent experience-induced cross-sensitization to Amph reward. [ABSTRACT FROM AUTHOR]

Abstract: Among 34 Streptococcus pneumoniae (Spn) sequential isolates from middle ear fluid, we found a case of a nontypeable S. pneumoniae (NT-Spn) in a child with acute otitis media (AOM). The strain was pneumolysin PCR positive and capsule gene PCR negative. Virulence of the NT-Spn was confirmed in a chinchilla model of AOM. [ABSTRACT FROM AUTHOR]

Abstract: Objective: Streptococcus pneumoniae (Spn) and Haemophilus influenzae (Hflu) are major etiologic pathogens for acute otitis media (AOM). However, when Spn and Hflu strains are not identified by traditional culture methods, use of alternative PCR-based diagnosis becomes critical. This study aimed to develop a combined molecular method to accurately detect these otopathogens. Methods: Middle ear fluid (MEF) samples were collected by tympanocentesis from children with AOM to isolate Spn and Hflu by standard culture procedures. Multiplex PCR (mPCR) and multi-locus sequence typing (MLST) techniques were used to detect Spn and Hflu in culture-negative MEF samples. Results: We found 20 Spn or Hflu culture-positive MEF samples that were mPCR-positive and typeable by MLST. The sequences of the housekeeping genes and the MLST allelic profiles obtained from Spn or Hflu culture isolates matched exactly MEF samples that were tested directly without culture isolation. Of 63 MEF samples that were culture-negative for Spn, 38% (24/63) were mPCR-positive for Spn. Of 50 MEF samples that were culture-negative for Hflu, 24% (12/50) were mPCR-positive for Hflu. Among these culture-negative but mPCR-positive MEF samples, 25% (6/24) and 25% (3/12) were typeable by MLST for Spn and Hflu, respectively. Conclusions: MEF samples may be analyzed with mPCR and MLST directly without culture isolation and the addition of mPCR and MLST may accurately identify Spn and Hflu in MEF of children with AOM when bacterial culture is negative. [ABSTRACT FROM AUTHOR]

Abstract: Development of natural antibodies to 3 nontypeable Haemophilus influenzae (NTHi) outer membrane proteins (D, P6 and OMP26) was prospectively studied in 130 children 6–30 months of age during NP colonization and acute otitis media (AOM). IgG antibody to protein D, P6 and OMP26 increased with age (p <0.001). Serum IgG responses to NP colonization were different for the 3 proteins: protein D responses occurred at a later age than P6, and OMP26 responses were minimal. For all 3 proteins serum antibody levels in the convalescent phase of AOM infection were not as high as after NP colonization. Antibodies to protein D and P6 but not OMP26 were bactericidal. [ABSTRACT FROM AUTHOR]

Examines the topology of the AE1 C-terminal region using cysteine-scanning mutagenesis and sulfhydryl-specific chemistry. Expression of individual cysteine residues by the transient transfection of HEK293 cells; Analysis of pertinent topics and relevant issues; Implications on lipids and lipoproteins.

Monocarboxylate transporters(MCTs)mediate the proton-coupled exchange of high-energy metabolites, including lactate and pyruvate, between cells and tissues. The transport activity of MCT1, MCT2, and MCT4 can be facilitated by the extracellular carbonic anhydrase IV (CAIV) via a noncatalytic mechanism. Combining physiological measurements in HEK-293 cells and Xenopus oocytes with pulldown experiments, we analyzed the direct interaction between CAIV and the two MCT chaperones basigin (CD147) and embigin (GP70). Our results show that facilitation ofMCTtransport activity requires direct binding ofCAIVto the transporters chaperones. We found that this binding is mediated by the highly conserved His-88 residue in CAIV, which is also the central residue of the enzyme's intramolecular proton shuttle, and a charged amino acid residue in the Ig1 domain of the chaperone. Although the position of the CAIV-binding site in the chaperone was conserved, the amino acid residue itself varied among different species. InhumanCD147, binding ofCAIVwas mediated by the negatively charged Glu-73 and in rat CD147 by the positively charged Lys-73. In rat GP70, we identified the positively charged Arg-130 as the binding site. Further analysis of the CAIV-binding site revealed that the His-88 inCAIVcan either act asHdonor orH acceptor for the hydrogen bond, depending on the charge of the binding residue in the chaperone. Our results suggest that the CAIV-mediated increase inMCTtransport activity requires direct binding between CAIV-His-88 and a charged amino acid in the extracellular domain of the transporter's chaperone. [ABSTRACT FROM AUTHOR]

Background There is no licensed vaccine for Moraxella catarrhalis ( Mcat ), which is a prominent bacterium causing acute otitis media (AOM) in children and lower respiratory tract infections in adults. Nasopharyngeal (NP) colonization caused by respiratory bacteria results in natural immunization of the host. To identify Mcat antigens as vaccine candidates, we evaluated the development of naturally induced antibodies to 5 Mcat surface proteins in children 6–30 months of age during Mcat NP colonization and AOM. Methods Human serum IgG against the recombinant Mcat proteins, outer membrane protein (OMP) CD, oligopeptide permease (Opp)A, hemagglutinin (Hag), Moraxella surface protein (Msp)22, and PilA clade 2 (PilA2) was quantitated by using an ELISA assay. Results There were 223 Mcat NP colonization episodes documented in 111 (60%) of 184 children in the study. Thirty five Mcat AOM episodes occurred in 30 (16%) of 184 children. All 5 Mcat candidate vaccine antigens evaluated stimulated a significant rise in serum IgG levles over time from 6 to 36 months of age ( P < 0.001), with a rank order as follows: Msp22 = OppA > OMP CD = Hag = PilA2. Children with no detectable Mcat NP colonization showed a higher serum IgG level against OppA, Hag, and Msp22 compared to those with Mcat NP colonization ( P < 0.05). Individual data showed that some children responded to AOM with an antibody increase to one or more of the studied Mcat proteins but some children failed to respond. Conclusions Serum antibody to Mcat candidate vaccine proteins OMP CD, OppA, Msp22, Hag, and PilA2 increased with age in naturally immunized children age 6–30 months following Mcat NP colonization and AOM. High antibody levels against OppA, Msp22, and Hag correlated with reduced carriage. The results support further investigation of these vaccine candidates in protecting against Mcat colonization and infection. [ABSTRACT FROM AUTHOR]

Human concentrative nucleoside transporter, hCNT3, mediates Na+/nucleoside and H+/nucleoside co-transport. We describe a new approach to monitor H+/uridine co-transport in cultured mammalian cells, using a pH-sensitive monomeric red fluorescent protein variant, mNectarine, whose development and characterization are also reported here. A chimeric protein, mNectarine fused to the N terminus of hCNT3 (mNect.hCNT3), enabled measurement of pH at the intracellular surface of hCNT3. mNectarine fluorescence was monitored in HEK293 cells expressing mNect.hCNT3 or mNect.hCNT3-F563C, an inactive hCNT3 mutant. Free cytosolic mNect, mNect.hCNT3, and the traditional pH-sensitive dye, BCECF, reported cytosotic pH similarly in pH-clamped HEK293 cells. Cells were incubated at the permissive pH for H+-coupled nucleoside transport, pH 5.5, under both Na+-free and Na+-containing conditions. In mNect.hCNT3-expressing cells (but not under negative control conditions) the rate of acidification increased in media containing 0.5 mM uridine, providing the first direct evidence for H+-coupled uridine transport. At pH 5.5, there was no significant difference in uridine transport rates (coupled H± flux) in the presence or absence of Na+ (1.09 ± 0.11 or 1.18 ± 0.32 mM min-1, respectively). This suggests that in acidic Na+-containing conditions, 1 Na+ and 1 H+ are transported per uridine molecule, while in acidic Na+-free conditions, 1 H+ alone is transported/uridine. In acid environments, including renal proximal tubule, H+/nucleoside co-transport may drive nucleoside accumulation by hCNT3. Fusion of mNect to hCNT3 provided a simple, self-referencing, and effective way to monitor nucleoside transport, suggesting an approach that may have applications in assays of transport activity of other H+-coupled transport proteins. [ABSTRACT FROM AUTHOR]

Kidney anion exchanger 1 (kAE1) mediates chloride/bicarbonate exchange at the basolateral membrane of kidney α-intercalated cells, thereby facilitating bicarbonate reabsorption into the blood. Human kAE1 lacks the N-terminal 65 residues of the erythroid form (AE1, band 3), which are essential for binding of cytoskeletal and cytosolic proteins. Yeast two-hybrid screening identified integrin-linked kinase (ILK), a serine/threonine kinase, and an actin-binding protein as an interacting partner with the N-terminal domain of kAE1. Interaction between kAE1 and ILK was confirmed in co-expression experiments in HEK 293 cells and is mediated by a previously unidentified calponin homology domain in the kAE1 N-terminal region. The calponin homology domain of kAE1 binds the C-terminal catalytic domain of ILK to enhance association of kAE1 with the actin cytoskeleton. Overexpression of ILK increased kAE1 levels at the cell surface as shown by flow cytometry, cell surface biotmylation, and anion transport activity assays. Pulse-chase experiments revealed that ILK associates with kAE1 early in biosynthesis, likely in the endoplasmic reticulum. ILK co-localized with kAE1 at the basolateral membrane of polarized Madin-Darby canine kidney cells and in α-intercalated cells of human kidneys. Taken together these results suggest that ILK and kAE1 traffic together from the endoplasmic reticulum to the basolateral membrane. ILK may provide a linkage between kAE1 and the underlying actin cytoskeleton to stabilize kAE1 at the basolateral membrane, resulting in higher levels of cell surface expression. [ABSTRACT FROM AUTHOR]

Abstract: Myocardial pHi recovery from intracellular alkalization results in part from the acid load (− J H+) carried by Cl−/HCO3 − anion-exchangers (AE). Three AE isoforms, AE1, AE2 and AE3, have been identified in cardiac membranes, but the function of each isoform on pHi homeostasis is still under investigation. This work explored, by means of specific antibodies, the role of AE3 isoform in myocardial pHi regulation. We developed rabbit polyclonal antibodies against the extracellular “loops”: one connecting the fifth to sixth and the other one the seventh to eighth transmembrane domains (loops 3 and 4, respectively) of AE3, and their effect on pHi regulation was studied in rat papillary muscles. The anti-AE3 loop 3 antibody decreased − J H+ in response to myocardial alkalization (from a mean control value of 1.06±0.26 to 0.32±0.13 mmol/L/min, n =7, P <0.05) without affecting the baseline pHi (7.22±0.03 vs. 7.21±0.04). The anti-AE3 loop 4 antibody did not modify either pHi recovery or baseline pHi. Under control conditions, endothelin-1 (ET-1) increased − J H+ in response to myocardial alkalization from 1.30±0.18 to 2.01±0.33 mmol/L /min (n =5, P <0.05). This effect of ET-1 on − J H+ was abolished by anti-AE3 loop 3 antibody. In addition, the MgATP-induced stimulation of AE activity was reduced by the anti-AE3 loop 3 antibody. These data support the key role of the AE3 isoform in myocardial pHi recovery from alkaline loads and also in the stimulatory effect of ET-1 on AE activity. To a lesser extent, it may also contribute to the effect of MgATP on pHi. [Copyright &y& Elsevier]

Over 200 million people worldwide are exposed to the human carcinogen, arsenic, in contaminated drinking water. In laboratory animals, arsenic and the essential trace element, selenium, can undergo mutual detoxification through the formation of the seleno-bis(S -glutathionyl) arsinium ion [(GS) 2 AsSe]−, which undergoes biliary and fecal elimination. [(GS) 2 AsSe]−, formed in animal red blood cells (RBCs), sequesters arsenic and selenium, and slows the distribution of both compounds to peripheral tissues susceptible to toxic effects. In human RBCs, the influence of arsenic on selenium accumulation, and vice versa, is largely unknown. The study aims were to characterize arsenite (AsIII) and selenite (SeIV) uptake by human RBCs, to determine if SeIV and AsIII increase the respective accumulation of the other in human RBCs, and ultimately to determine if this occurs through the formation and sequestration of [(GS) 2 AsSe]−. 75SeIV accumulation was temperature and Cl−-dependent, inhibited by 4,4′-diisothiocyanatodihydrostilbene-2,2′-disulfonic acid (H 2 DIDS) (IC 50 1 ± 0.2 µM), and approached saturation at 30 µM, suggesting uptake is mediated by the erythrocyte anion-exchanger 1 (AE1 or Band 3, gene SLC4A1). HEK293 cells overexpressing AE1 showed concentration-dependent 75SeIV uptake. 73AsIII uptake by human RBCs was temperature-dependent, partly reduced by aquaglyceroporin 3 inhibitors, and not saturated. AsIII increased 75SeIV accumulation (in the presence of albumin) and SeIV increased 73AsIII accumulation in human RBCs. Near-edge X-ray absorption spectroscopy revealed the formation of [(GS) 2 AsSe]− in human RBCs exposed to both AsIII and SeIV. The sequestration of [(GS) 2 AsSe]− in human RBCs potentially slows arsenic distribution to susceptible tissues and could reduce arsenic-induced disease. [ABSTRACT FROM AUTHOR]

Abstract: This work compares the design and practices for radiation safety among the five SR facilities: two low-energy sources (ALS and NSLS), one medium-energy source (SSRL), and two high-energy sources (APS and SPring8). The issues addressed in this comparison are (1) safety interlock systems for ring and beamlines, (2) beam loss scenarios and shielding design for storage ring, (3) beam loss scenarios and shielding design for SR beamlines, which cover synchrotron radiation and gas Bremsstrahlung issues, (4) radiation monitors for ring and beamlines, (5) safety control issues for top-up operation, and (6) operational issues. The goals of this work are to (1) provide a framework of radiation safety issues that need to, or may, be considered in the design and operation of a SR facility, and (2) develop sound policies and practices for radiation safety of SR facilities, when it is needed and practical to do so. [Copyright &y& Elsevier]