Checkpoint signaling, base excision repair, and PARP promote survival of colon cancer cells treated with 5-fluorodeoxyuridine but not 5-fluorouracil

The fluoropyrimidines 5-fluorouracil (5-FU) and FdUrd (5-fluorodeoxyuridine; floxuridine) are the backbone of chemotherapy regimens for colon cancer and other tumors. Despite their widespread use, it remains unclear how these agents kill tumor cells. Here, we have analyzed the checkpoint and DNA repair pathways that affect colon tumor responses to 5-FU and FdUrd. These studies demonstrate that both FdUrd and 5-FU activate the ATR and ATM checkpoint signaling pathways, indicating that they cause genotoxic damage. Notably, however, depletion of ATM or ATR does not sensitize colon cancer cells to 5-FU, whereas these checkpoint pathways promote the survival of cells treated with FdUrd, suggesting that FdUrd exerts cytotoxicity by disrupting DNA replication and/or inducing DNA damage, whereas 5-FU does not. We also found that disabling the base excision (BER) repair pathway by depleting XRCC1 or APE1 sensitized colon cancer cells to FdUrd but not 5-FU. Consistent with a role for the BER pathway, we show that small molecule poly(ADP-ribose) polymerase 1/2 (PARP) inhibitors, AZD2281 and ABT-888, remarkably sensitized both mismatch repair (MMR)-proficient and -deficient colon cancer cell lines to FdUrd but not to 5-FU. Taken together, these studies demonstrate that the roles of genotoxin-induced checkpoint signaling and DNA repair differ significantly for these agents and also suggest a novel approach to colon cancer therapy in which FdUrd is combined with a small molecule PARP inhibitor.     

Reversibility and binding kinetics of Thermobifida fusca cellulases studied through fluorescence recovery after photobleaching microscopy

Cellulases are enzymes capable of depolymerizing cellulose. Understanding their interactions with cellulose can improve biomass saccharification and enzyme recycling in biofuel production. This paper presents a study on binding and binding reversibility of Thermobifida fusca cellulases Cel5A, Cel6B, and Cel9A bound onto Bacterial Microcrystalline Cellulose. Cellulase binding was assessed through fluorescence recovery after photobleaching (FRAP) at 23, 34, and 45 °C. It was found that cellulase binding is only partially reversible. For processive cellulases Cel6B and Cel9A, an increase in temperature resulted in a decrease of the fraction of cellulases reversibly bound, while for endocellulase Cel5A this fraction remained constant. Kinetic parameters were obtained by fitting the FRAP curves to a binding-dominated model. The unbinding rate constants obtained for all temperatures were highest for Cel5A and lowest for Cel9A. The results presented demonstrate the usefulness of FRAP to access the fast binding kinetics characteristic of cellulases operating at their optimal temperature.     

Dynamics of fluorescence dequenching of ostrich-quenched fluorescein biotin: a multifunctional quantitative assay for biotin

We describe a simple and rapid quantitative assay for biotin and biotin conjugates. The assay is based on the kinetic analysis of the enhancement of fluorescence of streptavidin/fluorescein biotin complexes in the presence of biotin. The kinetic response of fluorescence enhancement is proportional to the concentration of biotin. Standard calibration curves based on the kinetic response are obtained and detection limits of approximately 10(-9)M are established. Because the assay is amenable for use in small volumes of 5-50 microL or bead-based assays, the detection limits can be extended to the femtomole range. Since the assay depends on kinetic analysis, routine quantitation can be achieved without reference to standard curves. The dynamic aspects allow the assay to be extended to a broader range of applications including its use as an indicator of reagent mixing in laminar-flow assays carried out in microfluidic devices.     

Push and pull of tropomyosin's opposite effects on myosin attachment to actin. A chimeric tropomyosin host-guest study

Tropomyosin is a ubiquitous actin-binding protein with an extended coiled-coil structure. Tropomyosin-actin interactions are weak and loosely specific, but they potently influence myosin. One such influence is inhibitory and is due to tropomyosin's statistically preferred positions on actin that sterically interfere with actin's strong attachment site for myosin. Contrastingly, tropomyosin's other influence is activating. It increases myosin's overall actin affinity ～4-fold. Stoichiometric considerations cause this activating effect to equate to an ～4(7)-fold effect of myosin on the actin affinity of tropomyosin. These positive, mutual, myosin-tropomyosin effects are absent if Saccharomyces cerevisiae tropomyosin replaces mammalian tropomyosin. To investigate these phenomena, chimeric tropomyosins were generated in which 38-residue muscle tropomyosin segments replaced a natural duplication within S. cerevisiae tropomyosin TPM1. Two such chimeric tropomyosins were sufficiently folded coiled coils to allow functional study. The two chimeras differed from TPM1 but in opposite ways. Consistent with steric interference, myosin greatly decreased the actin affinity of chimera 7, which contained muscle tropomyosin residues 228-265. On the other hand, myosin S1 increased by an order of magnitude the actin affinity of chimera 3, which contained muscle tropomyosin residues 74-111. Similarly, myosin S1-ADP binding to actin was strengthened 2-fold by substitution of chimera 3 tropomyosin for wild-type TPM1. Thus, a yeast tropomyosin was induced to mimic the activating behavior of mammalian tropomyosin by inserting a mammalian tropomyosin sequence. The data were not consistent with direct tropomyosin-myosin binding. Rather, they suggest an allosteric mechanism, in which myosin and tropomyosin share an effect on the actin filament.     

Ingestion of Salmonella enterica Serotype Poona by a Free-Living Nematode, Caenorhabditis elegans, and Protection against Inactivation by Produce Sanitizers

Free-living nematodes are known to ingest food-borne pathogens and may serve as vectors to contaminate preharvest fruits and vegetables. Caenorhabditis elegans was selected as a model to study the effectiveness of sanitizers in killing Salmonella enterica serotype Poona ingested by free-living nematodes. Aqueous suspensions of adult worms that had fed on S. enterica serotype Poona were treated with produce sanitizers. Treatment with 20 μg of free chlorine/ml significantly (α = 0.05) reduced the population of S. enterica serotype Poona compared to results for treating worms with water (control). However, there was no significant difference in the number of S. enterica serotype Poona cells surviving treatments with 20 to 500 μg of chlorine/ml, suggesting that reductions caused by treatment with 20 μg of chlorine/ml resulted from inactivation of S. enterica serotype Poona on the surface of C. elegans but not cells protected by the worm cuticle after ingestion. Treatment with Sanova (850 or 1,200 μg/ml), an acidified sodium chlorite sanitizer, caused reductions of 5.74 and 6.34 log₁₀ CFU/worm, respectively, compared to reductions from treating worms with water. Treatment with 20 or 40 μg of Tsunami 200/ml, a peroxyacetic acid-based sanitizer, resulted in reductions of 4.83 and 5.34 log₁₀ CFU/worm, respectively, compared to numbers detected on or in worms treated with water. Among the organic acids evaluated at a concentration of 2%, acetic acid was the least effective in killing S. enterica serotype Poona and lactic acid was the most effective. Treatment with up to 500 μg of chlorine/ml, 1% hydrogen peroxide, 2,550 μg of Sanova/ml, 40 μg of Tsunami 200/ml, or 2% acetic, citric, or lactic acid had no effect on the viability or reproductive behavior of C. elegans. Treatments were also applied to cantaloupe rind and lettuce inoculated with S. enterica serotype Poona or C. elegans that had ingested S. enterica serotype Poona. Protection of ingested S. enterica serotype Poona against sanitizers applied to cantaloupe was not evident; however, ingestion afforded protection of the pathogen on lettuce. These results indicate that S. enterica serotype Poona ingested by C. elegans may be protected against treatment with chlorine and other sanitizers, although the basis for this protection remains unclear.     

Role of claudin interactions in airway tight junctional permeability

Airway epithelial tight junctions (TJs) serve to separate the external and internal environments of the lung. However, the members of the claudin family that mediate this function have not been fully delineated. We characterized the claudin expression in normal airways removed from human donors during lung transplantation and determined the contribution of each claudin to airway barrier function. Stable cell lines in NIH/3T3 and human airway (IB3.1) cells were constructed expressing the claudin components found in the human airway, claudin-1, -3, or -5. The effects of claudin expression on transepithelial resistance, permeability coefficients, and claudin-claudin interactions were assessed. Claudin-1 and -3 decreased solute permeability, whereas claudin-5 increased permeability. We also detected oligomerization of claudin-5 in cell lines and in freshly excised human airways. Coimmunoprecipitation studies revealed heterophilic interactions between claudin species in both cell lines and human airway epithelium. These suggest that airway TJs are regulated by claudinclaudin interactions that confer the selectivity of the junction.     

Honey bee (Hymenoptera: Apidae) foraging in response to preconditioning with onion flower scent compounds

Onion (Allium cepa L.) seed production has long been plagued with yield problems because of lack of pollination by the honey bee, Apis mellifera L. To attempt to attract more pollinators to the onion seed production field, honey bees were conditioned to associate onion floral odor components with a reward. Isolated nucleus hives of honey bees were fed 30% sucrose solutions scented with a 0.2% solution of onion floral odor compounds. After feeding on these solutions for 6 wk, bees were not found to prefer onion flowers to two competing food sources, carrot and alfalfa flowers, at the 5% significance level. However, there was an overall trend indicating a change in honey bee behavior, with fewer "trained" bees visiting alfalfa and carrot and more visiting onion. Thus, it may be possible to alter honey bee behavior with preconditioning but probably not to a degree that would be economically significant.     

Hypertrophy, increased ejection fraction, and reduced Na-K-ATPase activity in phospholemman-deficient mice

Phospholemman (FXYD1), a 72-amino acid transmembrane protein abundantly expressed in the heart and skeletal muscle, is a major substrate for phosphorylation in the cardiomyocyte sarcolemma. Biochemical, cellular, and electrophysiological studies have suggested a number of possible roles for this protein, including ion channel modulator, taurine-release channel, Na(+)/Ca(2+) exchanger modulator, and Na-K-ATPase-associated subunit. We have generated a phospholemman-deficient mouse. The adult null mice exhibited increased cardiac mass, larger cardiomyocytes, and ejection fractions that were 9% higher by magnetic resonance imaging compared with wild-type animals. Notably, this occurred in the absence of hypertension. Total Na-K-ATPase activity was 50% lower in the phospholemman-deficient hearts. Expression (per unit of membrane protein) of total Na-K-ATPase was only slightly diminished, but expression of the minor alpha(2)-isoform, which has been specifically implicated in the control of contractility, was reduced by 60%. The absence of phospholemman thus results in a complex response, including a surprisingly large reduction in intrinsic Na-K-ATPase activity, changes in Na-K-ATPase isoform expression, increase in ejection fraction, and increase in cardiac mass. We hypothesize that a primary effect of phospholemman is to modulate the Na-K-ATPase and that its reduced activity initiates compensatory responses.     

Determinants of cardiac Na(+)/Ca(2+) exchanger temperature dependence: NH(2)-terminal transmembrane segments

The cardiacNa⁺/Ca²⁺ exchanger (NCX) in troutexhibits profoundly lower temperature sensitivity in comparison to themammalian NCX. In this study, we attempt to characterize the regions of the NCX molecule that are responsible for its temperature sensitivity. Chimeric NCX molecules were constructed using wild-type trout andcanine NCX cDNA and expressed in Xenopus oocytes.NCX-mediated currents were measured at 7, 14, and 30°C using thegiant excised-patch technique. By using this approach, the differentialtemperature dependence of NCX was found to reside within theNH₂-terminal region of the molecule. Specifically, we foundthat ~75% of the Na⁺/Ca²⁺ exchangedifferential energy of activation is attributable to sequencedifferences in the region that include the first four transmembranesegments, and the remainder is attributable to transmembrane segmentfive and the exchanger inhibitory peptide site.