Cystic Fibrosis Transmembrane Regulator-independent Release of ATP: ITS IMPLICATIONS FOR THE REGULATION OF P2Y2RECEPTORS IN AIRWAY EPITHELIA

The cystic fibrosis (CF) transmembrane regulator (CFTR) is a cyclic AMP-dependent Cl- channel that is defective in CF cells. It has been hypothesized that CFTR exhibits an ATP release function that controls the airway surface ATP concentrations. In airway epithelial cells, CFTR-independent Ca2+-activated Cl- conductance is regulated by the P2Y2 receptor. Thus, ATP may function as an autocrine signaling factor promoting Cl- secretion in normal but not CF epithelia if ATP release is defective. We have tested for CFTR-dependent ATP release using four independent detection systems. First, a luciferase assay detected no differences in ATP concentrations in the medium from control versus cyclic AMP-stimulated primary normal human nasal epithelial (HNE) cells. A marked accumulation of extracellular ATP resulted from mechanical stimulation effected by a medium displacement. Second, high pressure liquid chromatography analysis of 3H-labeled species released from [3H]adenine-loaded HNE cells revealed no differences between basal and cyclic AMP-stimulated cells. Mechanical stimulation of HNE cells again resulted in enhanced accumulation of extracellular [3H]ATP and [3H]ADP. Third, when measuring ATP concentrations via nucleoside diphosphokinase-catalyzed phosphorylation of [alpha-33P]dADP, equivalent formation of [33P]dATP was observed in the media of control and cyclic AMP-stimulated HNE cells and nasal epithelial cells from wild-type and CF mice. Mechanically stimulated [33P]dATP formation was similar in both cell types. Fourth, 1321N1 cells stably expressing the human P2Y2 receptor were used as a reporter system for detection of ATP via P2Y2 receptor-promoted formation of [3H]inositol phosphates. Basal [3H]inositol phosphate accumulation was of the same magnitude in control and CFTR-transduced cells, and no change was observed following addition of forskolin and isoproterenol. In both cell types, mechanical stimulation resulted in hexokinase-attenuable [3H]inositol phosphate formation. In summary, our data suggest that ATP release may be triggered by mechanical stimulation of cell surfaces. No evidence was found supporting a role for CFTR in the release of ATP

The Globular Cluster Systems in the Coma Ellipticals. II: Metallicity Distribution and Radial Structure in NGC 4874, and Implications for Galaxy Formation

Deep HST/WFPC2 (V,I) photometry is used to investigate the globular cluster system (GCS) in NGC 4874, the central cD galaxy of the Coma cluster. The luminosity function of the clusters displays its normal Gaussian-like shape and turnover level. Other features of the system are surprising: the GCS is (a) spatially extended, with core radius r_c = 22 kpc, (b) entirely metal-poor (a narrow, unimodal metallicity distribution with mean [Fe/H] = -1.5), and (c) modestly populated, with specific frequency S_N = 3.7 +- 0.5. We suggest on the basis of some simple models that as much as half of this galaxy might have accreted from low-mass satellites, but no single one of the three classic modes of galaxy formation (accretion, disk mergers, in situ formation) can supply a fully satisfactory formation picture. Even when they are used in combination, strong challenges to these models remain. The principal anomaly in this GCS is essentially the complete lack of metal-rich clusters. If these were present in normal (M87-like) numbers in addition to the metal-poor ones that are already there, then the GCS in total would more closely resemble what we see in many other giant E galaxies.Comment: 27 pp. with 9 Figures. Astrophys.J. 533, in press (April 10, 2000

Structure of Tagatose-1,6-bisphosphate Aldolase. Insight into chiral discrimination, mechanism, and specificity of class II aldolases

Tagatose-1,6-bisphosphate aldolase (TBPA) is a tetrameric class II aldolase that catalyzes the reversible condensation of dihydroxyacetone phosphate with glyceraldehyde 3-phosphate to produce tagatose 1,6-bisphosphate. The high resolution (1.45 Å) crystal structure of the Escherichia coli enzyme, encoded by the agaY gene, complexed with phosphoglycolohydroxamate (PGH) has been determined. Two subunits comprise the asymmetric unit, and a crystallographic 2-fold axis generates the functional tetramer. A complex network of hydrogen bonds position side chains in the active site that is occupied by two cations. An unusual Na(+) binding site is created using a interaction with Tyr(183) in addition to five oxygen ligands. The catalytic Zn(2+) is five-coordinate using three histidine nitrogens and two PGH oxygens. Comparisons of TBPA with the related fructose-1,6-bisphosphate aldolase (FBPA) identifies common features with implications for the mechanism. Because the major product of the condensation catalyzed by the enzymes differs in the chirality at a single position, models of FBPA and TBPA with their cognate bisphosphate products provide insight into chiral discrimination by these aldolases. The TBPA active site is more open on one side than FBPA, and this contributes to a less specific enzyme. The availability of more space and a wider range of aldehyde partners used by TBPA together with the highly specific nature of FBPA suggest that TBPA might be a preferred enzyme to modify for use in biotransformation chemistry

Naphthylisoquinoline alkaloids, validated as hit multistage antiplasmodial natural products

The discovery and development of multistage antimalarial drugs targeting intra-erythrocytic asexual and sexual Plasmodium falciparum parasites is of utmost importance to achieve the ambitious goal of malaria elimination. Here, we report the validation of naphthylisoquinoline (NIQ) alkaloids and their synthetic analogues as multistage active antimalarial drug candidates. A total of 30 compounds were tested, of which 17 exhibited IC50 values <1 μM against drug-sensitive P. falciparum parasites (NF54 strain); 15 of these retained activity against a panel of drug-resistant strains. These compounds showed low in vitro cytotoxicity against HepG2 cells, with selectivity indices of>10. The tested compounds showed activity in vitro against both early- and late-stage P. falciparum gametocytes while blocking male gamete formation (> 70% inhibition of exflagellation at 2 μM). Additionally, five selected compounds were found to have good solubility (≥170 μM in PBS at pH 6.5), while metabolic stability towards human, mouse, and rat microsomes ranged from>90% to >7% after 30 min. Dioncophylline C (2a) emerged as a front runner from the study, displaying activity against both asexual parasites and gametocytes, a lack of cross-resistance to chloroquine, good solubility, and microsomal stability. Overall, this is the first report on the multistage activity of NIQs and their synthetic analogues including gametocytocidal and gametocidal effects induced by this class of compounds.We thank Dr. Raina Seupel, Dr. Jun Li, Dr. Christine Schies, Prof. Dr. Dieudonné Tshitenge, and Dr. Sebastian Bischof for providing substances tested in this study. The UP ISMC acknowledges the SAMRC as Collaborating Centre for Malaria Research. This work was funded by the South African Research Chairs Initiative of the Department of Science and Technology, administered through the South African National Research Foundation to LMB (UID 84627), and supported by the SFB 630 “Agents against Infectious Diseases” funded by the Deutsche Forschungsgesellschaft (DFG) to GB. ACCdS and TJE acknowledge support of the National Institute of Allergy and Infectious Diseases of the National Institutes of Health under grant number R01AI143521. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health.https://www.elsevier.com/locate/ijpddram2020BiochemistryChemistryGeneticsMicrobiology and Plant Patholog

Genome-wide generation and systematic phenotyping of knockout mice reveals new roles for many genes.

Mutations in whole organisms are powerful ways of interrogating gene function in a realistic context. We describe a program, the Sanger Institute Mouse Genetics Project, that provides a step toward the aim of knocking out all genes and screening each line for a broad range of traits. We found that hitherto unpublished genes were as likely to reveal phenotypes as known genes, suggesting that novel genes represent a rich resource for investigating the molecular basis of disease. We found many unexpected phenotypes detected only because we screened for them, emphasizing the value of screening all mutants for a wide range of traits. Haploinsufficiency and pleiotropy were both surprisingly common. Forty-two percent of genes were essential for viability, and these were less likely to have a paralog and more likely to contribute to a protein complex than other genes. Phenotypic data and more than 900 mutants are openly available for further analysis. PAPERCLIP

Vanadium: a re-emerging environmental hazard

Vanadium (V) is a contaminant which has been long confined to the annals of regulatory history. This follows the reduction of its historical primary source (fossil fuel emissions) since the 1970s (e.g., by 80% in the UK). However, V is quickly becoming an important strategic resource which promises its return to environmental prominence because of changing industrial practices and emerging waste streams. We discuss below (i) what makes V a re-emerging environmental and human health hazard of global interest, (ii) the knowledge gaps that currently restrict prediction of environmental effect and mitigation, and (iii) opportunities for the community to address these gaps toward reducing the risk of an impending environmental hazard

Diversifying Selection Underlies the Origin of Allozyme Polymorphism at the Phosphoglucose Isomerase Locus in Tigriopus californicus

The marine copepod Tigriopus californicus lives in intertidal rock pools along the Pacific coast, where it exhibits strong, temporally stable population genetic structure. Previous allozyme surveys have found high frequency private alleles among neighboring subpopulations, indicating that there is limited genetic exchange between populations. Here we evaluate the factors responsible for the diversification and maintenance of alleles at the phosphoglucose isomerase (Pgi) locus by evaluating patterns of nucleotide variation underlying previously identified allozyme polymorphism. Copepods were sampled from eleven sites throughout California and Baja California, revealing deep genetic structure among populations as well as genetic variability within populations. Evidence of recombination is limited to the sample from Pescadero and there is no support for linkage disequilibrium across the Pgi locus. Neutrality tests and codon-based models of substitution suggest the action of natural selection due to elevated non-synonymous substitutions at a small number of sites in Pgi. Two sites are identified as the charge-changing residues underlying allozyme polymorphisms in T. californicus. A reanalysis of allozyme variation at several focal populations, spanning a period of 26 years and over 200 generations, shows that Pgi alleles are maintained without notable frequency changes. Our data suggest that diversifying selection accounted for the origin of Pgi allozymes, while McDonald-Kreitman tests and the temporal stability of private allozyme alleles suggests that balancing selection may be involved in the maintenance of amino acid polymorphisms within populations