Dark Matter Search Results from the PICO-2L C3F8 Bubble Chamber.

New data are reported from the operation of a 2 liter C3F8 bubble chamber in the SNOLAB underground laboratory, with a total exposure of 211.5 kg days at four different energy thresholds below 10 keV. These data show that C3F8 provides excellent electron-recoil and alpha rejection capabilities at very low thresholds. The chamber exhibits an electron-recoil sensitivity of <3.5×10(-10) and an alpha rejection factor of >98.2%. These data also include the first observation of a dependence of acoustic signal on alpha energy. Twelve single nuclear recoil event candidates were observed during the run. The candidate events exhibit timing characteristics that are not consistent with the hypothesis of a uniform time distribution, and no evidence for a dark matter signal is claimed. These data provide the most sensitive direct detection constraints on WIMP-proton spin-dependent scattering to date, with significant sensitivity at low WIMP masses for spin-independent WIMP-nucleon scattering.

Cost-free and sustainable incentive increases healthy eating decisions during elementary school lunch.

We aimed to develop a cost-free and sustainable program to influence healthier eating decisions during elementary school lunch. Baseline food and beverage choices were assessed for 9 days during lunch service at two racially and economically diverse elementary schools in Spartanburg County, SC, USA. After being informed that the labeled items on the daily lunch menu represented the healthiest choice, students were allowed to ring a call bell in the cafeteria for public recognition when they chose all of the identified healthiest food and beverage items during lunch service. Using menus matched to the baseline phase, food and beverage choices were measured during a 9-day intervention phase. After 30 days, food and beverage choices were reassessed during a 3-day follow-up phase. Healthiest food & beverage choices increased 49% with >60% of students choosing non-flavored milk over flavored milk during the intervention phase. There was no difference in the success of the program between the two schools. The program continued and healthy eating decisions were significantly sustained at a 30-day follow-up assessment. Public recognition through bell ringing appears to be an effective practice to sustain increases in healthy eating decisions during elementary school lunch and warrants expansion to larger scale, longitudinal trials.

Eight-month repellency and efficacy of an imidacloprid/flumethrin (Seresto® ) collar against the eastern paralysis tick (Ixodes holocyclus) on cats.

OBJECTIVE: The objective of this study was to determine the repellency and efficacy of a 10% imidacloprid/4.5% flumethrin (Seresto® , Elanco) collar over an 8-month period against the eastern paralysis tick (Ixodes holocyclus) on cats. METHODS: Two non-blinded, open gender, randomised, placebo-controlled pen studies were conducted, with 26 cats enrolled in each study. Prior to inclusion, cats were immunised with I. holocyclus holocyclotoxin. Cats were treated on Day 0 with either an imidacloprid/flumethrin or placebo collar. Tick infestations with 20 unfed adult female eastern paralysis ticks commenced on Day 7, and were repeated monthly for 8 months. Repellency was determined by comparing the mean number of attached ticks on imidacloprid/flumethrin treated cats, to placebo collar treated cats at 6 and 24 h post infestation. Efficacy was determined by comparing the mean number of live ticks on imidacloprid/flumethrin collar treated cats to placebo collar treated cats at 72 h post infestation. RESULTS: Efficacy was 100% (P < 0.001) at 72 h, and repellency was greater than 96% (P < 0.001) at 24 h for every tick challenge in each of the two studies, from Day 7 to the final infestation at 8 months for imidacloprid/flumethrin collar treated cats. CONCLUSIONS: In two pen studies, an imidacloprid/flumethrin collar controlled and repelled the eastern paralysis tick (I. holocyclus) on cats for 8-months. The marked repellency effect in addition to controlling tick paralysis would be beneficial in preventing tick bites and their sequelae.

Measurement of the ßß decay half-life of 130Te with the NEMO-3 detector.

We report results from the NEMO-3 experiment based on an exposure of 1275 days with 661 g of (130)Te in the form of enriched and natural tellurium foils. The ßß decay rate of (130)Te is found to be greater than zero with a significance of 7.7 standard deviations and the half-life is measured to be T(½)(2ν) = [7.0 ± 0.9(stat) ± 1.1(syst)] × 10(20) yr. This represents the most precise measurement of this half-life yet published and the first real-time observation of this decay.

Binding of the products of prothrombin activation to human platelets.

Binding of prothrombin and activation intermediates 1 and 2 to human platelets was tested with 125-I-labeled protein preparations. None of these precursors of thrombin bound to platelets under conditions in which high affinity binding of thrombin was observed,nor did they cause platelet aggregation or serotonin release. The molecular conformation required for binding to platelets as well as for induction of platelet aggregation and release is present, therefore, only after the final step in prothrombin activation.

Solution composition dependent variation in extinction coefficients for p-nitroaniline.

The dependence of the extinction coefficients for para-nitroaniline on solution composition has been investigated. The p-nitroaniline absorption spectrum is red-shifted with increasing ionic strength, with the consequence that the extinction coefficients at fixed wavelengths may vary significantly. The isosbestic wavelength for peptide p-nitroanilide/p-nitroaniline mixtures is similarly shifted. Poly(ethylene glycol) and bovine albumin, two additives frequently employed to eliminate enzyme loss from adsorption to cuvette and dilution vessel surfaces, also induce shifts in the p-nitroaniline spectrum. The use of a difference extinction coefficient at 381 nm, the p-nitroaniline maximum wavelength, is proposed to minimize the error resulting from solution composition dependent spectral shifts.

Effects of thermodynamic nonideality in kinetic studies: evidence of an expanded intermediate complex in the inactivation of thrombin by antithrombin III.

The technique of competitive chromogenic substrate hydrolysis is used to examine the inhibitory effects of sucrose and glycerol on the inactivation of thrombin by antithrombin III. This inhibition is attributed to the existence of a slight increase in volume/asymmetry associated with formation of the thrombin-antithrombin complex that subsequently undergoes covalent modification in an irreversible inactivation step. Partial reversal of the equilibrium step is thus considered to result from the effects of molecular crowding in the highly concentrated environment that is generated by the inclusion of these small insert solutes.

Measurement of phospholipid monolayer surface potentials at a hydrocarbon-electrolyte interface.

The phospholipid monolayer spread at a hydrocarbon-electrolyte interface can be used as a model system for the plasma membrane and its properties and structure probed by measurements of surface pressure and surface potential. To facilitate such studies, (i) the theory of the vibrating plate (Kelvin) method of measuring surface potentials is reëxamined and a new interpretation given for the potentials measured and (ii) a new apparatus for performing these measurements is described. The theory and apparatus are illustrated by measurements on films of distearoyl phosphatidylcholine at the interface between 2,2,4-trimethylpentane (isooctane) and 0.1 M NaCl.

Phospholipid monolayers at the hydrocarbon-electrolyte interface. The interrelation of film potential and film pressure.

Measurements of surface pressure of surface potential are reported for films of distearoyl phosphatidylcholine (density range: 0.15--2.65 . 10(18) molecules/m2) spread at the interface between 2,2,4-trimethylpentane and 100 mM NaCl. Low density behavior of the surface pressure is explained using classical viral theory. The behavior of the surface potential is qualitatively explained for all densities in terms of the dipole moments associated with the carboxyl groups and headgroups of the phosphatidylcholine.

The action of factor Xa on peptide p-nitroanilide substrates: substrate selectivity and examination of hydrolysis with different reaction conditions.

Kinetic parameters for the action of bovine Factor Xa (EC 3.4.21.22) on 25 commercially available peptide p-nitroanilides have been determined. The selectivity constant, kc/Km, ranges from 1.5 X 10(1) to 2 X 10(6) M-1 X s-1 for the poorest and the best substates, respectively. The best substrates for Factor Xa were identified as those with arginine in the P1 position, and glycine in the P2 position. Quantitative distinction between lysine and arginine in the P1 position and other amino acids in the P2-P4 positions of the substrate is reported from the changes in the kinetic parameters for substrates differing in only a single amino acid in these positions. Effect of NaCl and CaCl2 concentrations and temperature on the action of Factor Xa on selected substrates have been assessed. Km values for Factor Xa hydrolysis of most substrates are greater than 100 microM. Solubility of the substrates consequently restricts measurements of reaction velocities to concentrations lower than desirable for optimally determining kc. Comparison of these kinetic parameters for Factor Xa with those of thrombin (Lottenberg, R., Hall, J.A., Blinder, M., Binder, E. and Jackson, C.M. (1983) Biochim. Biophys. Acta 742,539-557) for these same substrates indicates that the greater hydrolytic efficiency of thrombin is due primarily to lower Km values.

The action of thrombin on peptide p-nitroanilide substrates. Substrate selectivity and examination of hydrolysis under different reaction conditions.

Kinetic parameters for the action of bovine alpha-thrombin on 24 commercially available peptide p-nitroanilides have been determined. The selectivity constant, kcat/Km, ranges from 3.3 X 10(1) to 1.1 X 10(8) M-1 X S-1 for the poorest and the best substrates, respectively. The best substrates for thrombin were identified as those with arginine in the P1 position, proline or a proline homolog in the P2 position, and an apolar amino acid in the P3 position. Quantitative distinction between lysine and arginine in the P1 position and other amino acids in the P2-P4 positions of the substrate is reported from the changes in the kinetic parameters for substrates differing in only a single amino acid in these positions. Effects of NaCl, CaCl2 and poly(ethylene glycol) concentrations, pH and temperature on the action of thrombin on selected substrates have been assessed. A source of large systematic error in thrombin concentration estimates was identified as resulting from adsorption losses. These losses were eliminated by inclusion of poly(ethylene glycol) in dilution and reaction buffers.

A kinetic model describing the interaction of bovine prothrombin fragment 1 with calcium ions.

A kinetic model is derived for the interaction of bovine prothrombin fragment 1 with calcium ions. The model requires binding of a minimum of two calcium ions for induction of the observed biphasic fluorescence decrease as a function of time. The model is shown to be consistent with experimental kinetic and equilibrium data by fitting theoretical curves for the biphasic fluorescence change to the data through exact solution of the nonlinear differential rate equations derived from the model. The rate constants for the binding of these two required calcium ions are calculated from the solutions as best fit parameters. The thermodynamic equilibrium constants, K1 and K2, for the binding of these two calcium ions are calculated from ratios of the forward and reverse rate constants as 0.6 X 10(4) and 5.4 X 10(4), respectively. Thus, the model correctly predicts positively cooperative calcium ion binding for at least the two calcium ions required to induce fluorescence quenching.

Characteristics of cyanopindolol analogues active at the beta 3-adrenoceptor in rat ileum.

1. Cyanopindolol (CYP) is a potent antagonist at the beta 3-adrenoceptor in rat ileum. Several analogues of CYP and pindolol were synthesized that also produced antagonist effects at the beta 3-adrenoceptor. However, at high concentrations, these compounds appear to act as "partial agonists'. This study was conducted to determine the structural requirements of CYP analogues necessary for antagonist activity and to examine the possibility that the agonist effects of CYP and its analogues may occur through a mechanism independent of beta-adrenoceptor activation. 2. Analogues of CYP and pindolol were tested for antagonist activity in rat ileum in which the beta 1- and beta 2-adrenoceptors were blocked. Fourteen compounds were tested against (-)-isoprenaline, and four of the more potent analogues were then tested against BRL 37344. The two most potent antagonists were CYP and iodocyanopindolol. The pKb values (negative log of equilibrium dissociation constant) obtained against (-)-isoprenaline were significantly higher than those obtained against BRL 37344, but the cause of this difference is not known. 3. Several structural requirements were determined for antagonist activity. Modification at the carbon atom alpha to the secondary amine caused the antagonist potency to fall as the level of saturation was reduced. Thus, a quaternary carbon group, such as t-butyl, produced the most potent antagonist. Substitution with a large moiety such as a cyclohexyl or benzyl group reduced antagonist activity, probably due to steric hindrance. Inclusion of an electron-withdrawing group, such as a cyano or ethylester moiety, alpha to the indole nitrogen, also increased the potency. Iodination of CYP and ethylesterpindolol at the 3-position of the indole ring did not increase antagonist potency. In contrast, iodination of the almost inactive analogues produced a significant increase in potency, suggesting that a beneficial electronic effect on the indole ring imparted by the iodo moiety may be able to offset partially the negative effects caused by either the steric hindrance, of lack of a quaternary carbon alpha to the secondary amine. 4. Values for pseudo-pD2 were also determined by conducting cumulative concentration-response studies up to the limit of drug solubility. For nine of the compounds tested, the pKb was significantly higher than the pseudo-pD2 value. 5. The discrepancy between the pKb and pseudo-pD2 values was examined further. The agonist effects of iodocyanopindolol, the agonist with the highest potency, were not antagonized by CYP which was the most potent antagonist of (-)-isoprenaline and BRL 37344 at the beta 3-adrenoceptor. This suggests that the agonist effects of iodoCYP were produced through a different mechanism: either via another receptor, another isoform of the rat beta 3-adrenoceptor, or through a non-receptor-mediated effect. Pseudo-pD2 values did not correlate with log P values for these compounds, indicating that their relaxant effects were not simply a function of their lipid solubility. 6. This study has highlighted several structural requirements for antagonist binding potency at the rat ileum beta 3-adrenoceptor and should assist in the development of potent selective antagonists for this receptor.

Calcium ion-protein interactions in prothrombin activation.

1. The protein concentration dependence observed in the calcium binding to fragment 1 indicates that calcium-mediated dimerization is responsible for the cooperative calcium binding behavior usually observed. "Unusual" fragment 1, which exhibits negative cooperativity (the type of binding behavior expected for ions interacting with a charged protein) at high concentration, also exhibit altered self-association behavior. 2. The calcium-induced spectral perturbations that are observed by fluorescence and ultraviolet difference spectroscopy are influenced by calcium-mediated dimerization. Similar spectral perturbations may also be induced by other divalent, trivalent, and monovalent ions, as well as changes in pH. Because this is a multi-site system, only limited interpretation of the spectral data is possible without calcium binding data. 3. Although strong side chain CD signals make estimation of fragment 1 secondary structure ambiguous, the CD data do indicate small changes in structure during calcium binding. Similar changes are observed upon addition of monovalent ions at high concentration or after lowering the pH. No coupling between changes in conformation and the cooperative calcium binding behavior has yet been observed to exist.

Airway-stabilizing effect of long-acting beta2-agonists as add-on therapy to inhaled corticosteroids.

BACKGROUND: The protection afforded by long-acting beta(2)-agonists against bronchoconstrictor stimuli can be regarded as a surrogate for their stabilizing effects on airway smooth muscle. AIM: To determine the magnitude of residual bronchoprotection after chronic dosing with long-acting beta(2)-agonists. DESIGN: Retrospective meta-analysis. METHODS: Medline, BIDS and Cochrane Library databases were searched from 1990. A meta-analysis was then performed of 13 eligible randomized placebo-controlled trials (596 patients) in which second-line treatment with a long-acting beta(2)-agonist (salmeterol or formoterol) was used for 1 week or more. The residual protection against bronchoconstrictor stimuli as doubling dose/dilution shift was the main outcome measure. RESULTS: Data were assessed according to Quorum criteria. Combining the results of the meta-analysis, the overall estimated protection amounted to a 0.79 (95%CI 0.63-0.96) doubling dose/dilution shift from placebo. Subgroup analysis showed greater protection at peak vs. trough, but no difference between formoterol vs. salmeterol, or between direct vs. indirect challenge. There was no evidence of significant heterogeneity across all the studies, or within any of the subgroups. DISCUSSION: When used as second-line treatment, the overall additive protective effect of long-acting beta(2)-agonists amounts to a 0.8 doubling dose/dilution shift. This stabilizing effect on airway smooth muscle may explain their beneficial effects on exacerbations.

Safety of inhaled and intranasal corticosteroids: lessons for the new millennium.

Although inhaled and intranasal corticosteroids are first-line therapy for asthma and allergic rhinitis, there has recently been an increasing awareness of their propensity to produce systemic adverse effects. The availability of more potent and lipophilic corticosteroids and new chlorofluorocarbon (CFC)-free formulations has focused attention on these safety issues. The main determinant of systemic bioavailability of these drugs is direct absorption from the lung or nose, where there is no first-pass inactivation. Consequently, the systemic bioavailability of inhaled corticosteroids is greatly influenced by the efficiency of the inhaler device. Thus, when comparing different inhaled corticosteroids it is imperative to consider the unique drug/device interaction. The pharmacokinetic profile is important in determining the systemic bioactivity of inhaled and intranasal corticosteroids. For highly lipophilic drugs, such as fluticasone propionate or mometasone furoate, there is preferential partitioning into the systemic tissue compartment, and consequently a large volume of distribution at steady state. In contrast, drugs with lower lipophilicity, such as triamcinolone acetonide or budesonide, have a smaller volume of distribution. The systemic tissue compartment may act as a slow release reservoir, resulting in a long elimination half-life for the lipophilic drugs. For intranasal corticosteroids, a high degree of lipophilicity diminishes water solubility in mucosa and therefore increases the amount of drug swept away by mucociliary clearance before it can gain access to tissue receptor sites. This may reduce the anti-inflammatory efficacy in the nose, but might also reduce the propensity for direct systemic absorption from the nasal cavity. The hydrofluoroalkane (HFA) formulations of beclomethasone dipropionate are solutions and exhibit a much higher respirable fine particle dose than do the CFC formulations. Dose-response studies with one of the HFA formulations have shown therapeutic equivalence at half the dosage, with little evidence of adrenal suppression at dosages up to 800 microg/day. A lack of similar studies for another of the available HFA formulations has led to a discrepancy in the recommendations for equivalence. Although in vitro studies have pointed to a similar fine particle distribution for the HFA and CFC formulations of fluticasone propionate, this is not supported by in vivo data for lung bioavailability, suggesting that care will be required when switching these formulations. Prescribers of inhaled and intranasal corticosteroids should be aware of the potential for long term systemic effects. The safest way to use these drugs is to 'step-down' to achieve the lowest possible effective maintenance dosage.

Comparison of methods for detecting specific IgM antibody in infants with congenital rubella.

Serum specimens from 14 infants with congenital rubella were examined for specific IgM antibody by six different methods. IgM-containing fractions were separated either by sucrose density-gradient centrifugation or by gel filtration through Sephadex G-200, and were then tested by the indirect immunofluorescence technique and by the haemagglutination-inhibition (HI) test (long-and short-incubation methods). Immunofluorescence staining of density-gradient fractions detected specific IgM in all 14 infants. The HI test (long method), applied to density-gradient fractions, was almost as sensitive, detecting antibody in 13 infants; the short method was less sensitive. The gel-filtration technique proved to be generally less satisfactory than sucrose density-gradient centrifugation. Evidence was obtained for the occurrence of as yet unclassified non-specific inhibitors in the serum of some infants. These inhibitors were deposited with the IgM on sucrose-density gradients and they could have been mistaken for rubella-specific IgM antibody, particularly in the HI test (long method).

Reaction of thrombins with human antithrombin III: II. Dependence of rate of inhibition on molecular form and origin of thrombin.

The rate of thrombin inhibition by AT III depends upon the molecular form (alpha, beta, gamma) and species origin of the enzyme. The following apparent second order rate constants (.1000/M.s) were established alpha human 11.24 +/- 0.8; alpha bovine 7.46 +/- 0.27; beta bovine 6.49 +/- 0.34 and gamma human thrombin 2.80 +/- 0.11, 25 degrees C, pH = 7.80, 0.01 M TRIS, 0.01 M HEPES buffer, 0.0025 M EDTA, 0.3 M NaCl, 1 mg/mL PEG 6000. Using these values, the concentration of active AT III in an unknown sample can be calculated from the measured apparent first order rate constant in moles/liter instead of relative units. In contrast to the reactions in the absence of heparin, in the presence of high affinity heparin, the differences between various forms of thrombin are more pronounced and the shape of the progress curves, as well as rates, are highly dependent on the ionic strength. In the presence of heparin, measurement of the rate of inhibition under pseudo first order conditions can be made only when the NaCl concentration is at least 0.3 M. The significance of the presented data for designing a functional assay of AT III is discussed.

The action of thrombin on peptide p-nitroanilide substrates: hydrolysis of Tos-Gly-Pro-Arg-pNA and D-Phe-Pip-Arg-pNA by human alpha and gamma and bovine alpha and beta-thrombins.

Human and bovine alpha-thrombins (greater than 90% alpha form) with high fibrinogen clotting activities (approximately 3,000 U.S. units/mg protein) exhibit similar Michaelis menten kinetics with the p-nitroanilide tripeptide substrates Tos-Gly-Pro-arg-pNA (Chromozym-TH) and D-Phe-Pip-Arg-pNA (S-2238). The kinetic parameters at I = 0.11 M, 25 degrees C, pH 7.8 are: (Km = 4.18 +/- 0.22 and 3.61 +/- 0.15 microM; kcat = 127 +/- 8 and 100 +/- 1 s-1) for Chromozym TH and (Km = 1.33 +/- 0.07 and 1.50 +/- 0.10 microM; kcat = 91.4 +/- 1.8 and 98.0 +/- 0.5 s-1) for S-2238 for the human and bovine enzymes, respectively. Unlike the native enzyme forms, their "non-clotting" terminal degradative forms, human gamma-thrombin (approximately 5 units/mg) and bovine beta-thrombin (approximately 200 units/mg), give increased values for these parameters (km = 14.3 +/- 2.4 and 14.4 +/- 2.2 microM; kcat = 160 +/- 9 and 124 +/- 6 s-1) for Chromozym-TH; and (Km = 2.50 +/- 0.36 and 2.99 +/- 0.33 microM; kcat = 106 +/- 3 and 106 +/- 3 s-1) for S-2238. Based on these parameters, 50% degradation of human or bovine alpha-thrombins can be calculated to produce relatively small errors in the kinetic measurement of total thrombin concentrations (maximally 9% and 7% for Chromozym-TH; 7% and 3% for S-2238, respectively) if the kinetic parameters for all alpha forms are erroneously used and assays are at 150 microM substrate. This is in contrast to the large errors inherent in clotting activity measurements on thrombin mixtures. Incorporation of 1 mg/ml of polyethylene glycol 6,000 into assay solutions eliminates systematic errors otherwise caused by thrombin adsorption to surfaces and enables thrombin to be accurately assayed at concentrations less than 0.1 nM or 0.01 clotting unit/ml of alpha-thrombin.

Evidence for multiple enzyme site involvement in the modulation of thrombin activity by products of prothrombin proteolysis.

Kinetic evidence is presented for the interaction of prothrombin with several distinctive topological regions of the thrombin molecule. Modulations of thrombin catalytic activity on the protein substrates prothrombin and prethrombin 1 are demonstrated that involve the fragment 1 and fragment 2 portions. The inhibitory effects are demonstrably non-competitive. In addition to exhibiting non-competitive inhibition, fragment 2 is capable of enhancing proteolysis by thrombin; and therefore to react with a second region of the enzyme. On the basis of the crystallographic studies of the complex between fragment 2 and thrombin (Arni et al., Biochemistry 32 (1992) 4727), this activating site is proposed to be associated with exosite II. The allosteric switch between procoagulant and anticoagulant activities identified from studies by Di Cera (Dang et al., Proc. Natl. Acad. Sci USA 92 (1995) 5977) could be 'thrown' by a macromolecular effector that is generated during thrombin formation--a plausible mechanism for switching that deserves further investigation.