MiniBooNE and MicroBooNE Combined Fit to a 3+1 Sterile Neutrino Scenario.

This Letter presents the results from the MiniBooNE experiment within a full "3+1" scenario where one sterile neutrino is introduced to the three-active-neutrino picture. In addition to electron-neutrino appearance at short baselines, this scenario also allows for disappearance of the muon-neutrino and electron-neutrino fluxes in the Booster Neutrino Beam, which is shared by the MicroBooNE experiment. We present the 3+1 fit to the MiniBooNE electron-(anti)neutrino and muon-(anti)neutrino data alone and in combination with MicroBooNE electron-neutrino data. The best-fit parameters of the combined fit with the exclusive charged-current quasielastic analysis (inclusive analysis) are Δm^{2}=0.209 eV^{2}(0.033 eV^{2}), |U_{e4}|^{2}=0.016(0.500), |U_{µ4}|^{2}=0.500(0.500), and sin^{2}(2θ_{µe})=0.0316(1.0). Comparing the no-oscillation scenario to the 3+1 model, the data prefer the 3+1 model with a Δχ^{2}/d.o.f.=24.7/3(17.3/3), a 4.3σ(3.4σ) preference assuming the asymptotic approximation given by Wilks's theorem.

Significant Excess of Electronlike Events in the MiniBooNE Short-Baseline Neutrino Experiment.

The MiniBooNE experiment at Fermilab reports results from an analysis of ν_{e} appearance data from 12.84×10^{20} protons on target in neutrino mode, an increase of approximately a factor of 2 over previously reported results. A ν_{e} charged-current quasielastic event excess of 381.2±85.2 events (4.5σ) is observed in the energy range 200

First Measurement of Monoenergetic Muon Neutrino Charged Current Interactions.

We report the first measurement of monoenergetic muon neutrino charged current interactions. MiniBooNE has isolated 236 MeV muon neutrino events originating from charged kaon decay at rest (K^{+}→µ^{+}ν_{µ}) at the NuMI beamline absorber. These signal ν_{µ}-carbon events are distinguished from primarily pion decay in flight ν_{µ} and ν[over ¯]_{µ} backgrounds produced at the target station and decay pipe using their arrival time and reconstructed muon energy. The significance of the signal observation is at the 3.9σ level. The muon kinetic energy, neutrino-nucleus energy transfer (ω=E_{ν}-E_{µ}), and total cross section for these events are extracted. This result is the first known-energy, weak-interaction-only probe of the nucleus to yield a measurement of ω using neutrinos, a quantity thus far only accessible through electron scattering.

Prognostic factors for survival after complete resections of synchronous lung cancers in multiple lobes: pooled analysis based on individual patient data.

BACKGROUND: Some reports suggest that patients with synchronous multiple foci of nonsmall-cell lung cancers (NSCLC) distributed in multiple lobes have a poor prognosis, even when there is no extrathoracic metastasis. The vast majority of such patients do not receive surgical treatment. For those who undergo surgery, prognostic factors are unclear. PATIENTS AND METHODS: We systematically reviewed the literature on surgery for synchronous NSCLC in multiple lobes published between 1990 and 2011. Individual patient data were used to obtain adjusted hazard ratios (HRs) in each dataset and pooled analyses were carried out. RESULTS: Six studies contributed 467 eligible patients for analysis. The median overall survival was 52.0 months [95% confidence interval 45.6-63.7]. Male gender and advanced age were associated with a decreased survival: HRs 1.64 (1.22, 2.22) and 1.40 (1.20, 1.80) per 20-year increment, respectively. Patients with cancers distributed in one lung had a higher mortality risk than those with bilateral disease: HRs 1.45 (1.06, 2.00). N1 or N2 had a decreased survival compared with N0: HRs 1.68 (1.12, 2.51) and 1.94 (1.33, 2.82), respectively. There was a trend toward increased mortality among patients with different histology: HRs 1.29 (0.96, 1.75). CONCLUSION: Advanced age, male gender, nodal involvement, and unilateral tumor location were poor prognostic factors.

Improved search for ν¯(µ)→ν¯(e) oscillations in the MiniBooNE experiment.

The MiniBooNE experiment at Fermilab reports results from an analysis of ν[over ¯](e) appearance data from 11.27×10(20) protons on target in the antineutrino mode, an increase of approximately a factor of 2 over the previously reported results. An event excess of 78.4±28.5 events (2.8σ) is observed in the energy range 200

The base of the proteasome regulatory particle exhibits chaperone-like activity.

Protein substrates of the proteasome must apparently be unfolded and translocated through a narrow channel to gain access to the proteolytic active sites of the enzyme. Protein folding in vivo is mediated by molecular chaperones. Here, to test for chaperone activity of the proteasome, we assay the reactivation of denatured citrate synthase. Both human and yeast proteasomes stimulate the recovery of the native structure of citrate synthase. We map this chaperone-like activity to the base of the regulatory particle of the proteasome, that is, to the ATPase-containing assembly located at the substrate-entry ports of the channel. Denatured but not native citrate synthase is bound by the base complex. Ubiquitination of citrate synthase is not required for its binding or refolding by the base complex of the proteasome. These data suggest a model in which ubiquitin-protein conjugates are initially tethered to the proteasome by specific recognition of their ubiquitin chains; this step is followed by a nonspecific interaction between the base and the target protein, which promotes substrate unfolding and translocation.

In vivo half-life of a protein is a function of its amino-terminal residue.

When a chimeric gene encoding a ubiquitin-beta-galactosidase fusion protein is expressed in the yeast Saccharomyces cerevisiae, ubiquitin is cleaved off the nascent fusion protein, yielding a deubiquitinated beta-galactosidase (beta gal). With one exception, this cleavage takes place regardless of the nature of the amino acid residue of beta gal at the ubiquitin-beta gal junction, thereby making it possible to expose different residues at the amino-termini of the otherwise identical beta gal proteins. The beta gal proteins thus designed have strikingly different half-lives in vivo, from more than 20 hours to less than 3 minutes, depending on the nature of the amino acid at the amino-terminus of beta gal. The set of individual amino acids can thus be ordered with respect to the half-lives that they confer on beta gal when present at its amino-terminus (the "N-end rule"). The currently known amino-terminal residues in long-lived, noncompartmentalized intracellular proteins from both prokaryotes and eukaryotes belong exclusively to the stabilizing class as predicted by the N-end rule. The function of the previously described posttranslational addition of single amino acids to protein amino-termini may also be accounted for by the N-end rule. Thus the recognition of an amino-terminal residue in a protein may mediate both the metabolic stability of the protein and the potential for regulation of its stability.

Proteolysis. The proteasome: a protein-degrading organelle?

The crystal structure of the proteasome suggests that degradation of ubiquitin-protein conjugates is achieved by unfolding the protein substrate and translocating it through a channel into a peptidase-containing chamber.

Synthetic signals for ubiquitin-dependent proteolysis.

Short-lived proteins are targeted for turnover by sequence elements known as degradation signals. Because of the large size and heterogeneity of these signals, the structural features important for their function are not well defined. In this study, we have isolated three classes of degradation signals by screening short artificial sequences for the ability to destabilize a reporter protein. Class I and class II signals were derived by inserting random nonapeptide sequences after the second residue of beta-galactosidase. Class III signals contained five-residue homopolymers at the same position. Class I beta-galactosidase turnover was inhibited in mutants lacking either the ubiquitin-conjugating enzyme Ubc2 or the ubiquitin protein ligase Ubr1. Class I random inserts functioned to promote N-terminal proteolytic processing and define a novel pathway for exposure of residues that are destabilizing according to the N-end rule. Efficient degradation of proteins containing class II signals required at least three Ubc enzymes: Ubc6, Ubc7, and either one of the related enzymes Ubc4 and Ubc5. Analysis of 56 amino acid substitutions in the class II signal suggested that it is recognized in the form of an amphipathic alpha helix. Class III signals consisted of short tracts of hydrophobic residues such as Leu and Ile. Degradation of class III proteins involved the Ubc4 and Ubc5 enzymes but not Ubc2, Ubc6, or Ubc7. Clusters of hydrophobic residues appear to be critical for the recognition of both class II and class III signals.

A conserved sequence in histone H2A which is a ubiquitination site in higher eucaryotes is not required for growth in Saccharomyces cerevisiae.

Histones H2A and H2B are modified by ubiquitination of specific lysine residues in higher and lower eucaryotes. To identify functions of ubiquitinated histone H2A, we studied an organism in which genetic analysis of histones is feasible, the yeast Saccharomyces cerevisiae. Surprisingly, immunoblotting experiments using both anti-ubiquitin and anti-H2A antibodies gave no evidence that S. cerevisiae contains ubiquitinated histone H2A. The immunoblot detected a variety of other ubiquitinated species. A sequence of five residues in S. cerevisiae histone H2A that is identical to the site of H2A ubiquitination in higher eucaryotes was mutated to substitute arginines for lysines. Any ubiquitination at this site would be prevented by these mutations. Yeast organisms carrying this mutation were indistinguishable from the wild type under a variety of conditions. Thus, despite the existence in S. cerevisiae of several gene products, such as RAD6 and CDC34, which are capable of ubiquitinating histone H2A in vitro, ubiquitinated histone H2A is either scarce in or absent from S. cerevisiae. Furthermore, the histone H2A sequence which serves as a ubiquitination site in higher eucaryotes is not essential for yeast growth, sporulation, or resistance to either heat stress or UV radiation.

A ubiquitin mutant with specific defects in DNA repair and multiubiquitination.

The degradation of many proteins involves the sequential ligation of ubiquitin molecules to the substrate to form a multiubiquitin chain linked through Lys-48 of ubiquitin. To test for the existence of alternate forms of multiubiquitin chains, we examined the effects of individually substituting each of six other Lys residues in ubiquitin with Arg. Substitution of Lys-63 resulted in the disappearance of a family of abundant multiubiquitin-protein conjugates. The UbK63R mutants were not generally impaired in ubiquitination, because they grew at a wild-type rate, were fully proficient in the turnover of a variety of short-lived proteins, and exhibited normal levels of many ubiquitin-protein conjugates. The UbK63R mutation also conferred sensitivity to the DNA-damaging agents methyl methanesulfonate and UV as well as a deficiency in DNA damage-induced mutagenesis. Induced mutagenesis is mediated by a repair pathway that requires Rad6 (Ubc2), a ubiquitin-conjugating enzyme. Thus, the UbK63R mutant appears to be deficient in the Rad6 pathway of DNA repair. However, the UbK63R mutation behaves as a partial suppressor of a rad6 deletion mutation, indicating that an effect of UbK63R on repair can be manifest in the absence of the Rad6 gene product. The UbK63R mutation may therefore define a new role of ubiquitin in DNA repair. The results of this study suggest that Lys-63 is used as a linkage site in the formation of novel multiubiquitin chain structures that play an important role in DNA repair.

The molecular chaperone Ydj1 is required for the p34CDC28-dependent phosphorylation of the cyclin Cln3 that signals its degradation.

The G1 cyclin Cln3 of the yeast Saccharomyces cerevisiae is rapidly degraded by the ubiquitin-proteasome pathway. This process is triggered by p34CDC28-dependent phosphorylation of Cln3. Here we demonstrate that the molecular chaperone Ydj1, a DnaJ homolog, is required for this phosphorylation. In a ydj1 mutant at the nonpermissive temperature, both phosphorylation and degradation of Cln3 were deficient. No change was seen upon inactivation of Sis1, another DnaJ homolog. The phosphorylation defect in the ydj1 mutant was specific to Cln3, because no reduction in the phosphorylation of Cln2 or histone H1, which also requires p34CDC28, was observed. Ydj1 was required for Cln3 phosphorylation and degradation rather than for the proper folding of this cyclin, since Cln3 produced in the ydj1 mutant was fully active in the stimulation of p34CDC28 histone kinase activity. Moreover, Ydj1 directly associates with Cln3 in close proximity to the segment that is phosphorylated and signals degradation. Thus, binding of Ydj1 to this domain of Cln3 seems to be essential for the phosphorylation and breakdown of this cyclin. In a cell-free system, purified Ydj1 stimulated the p34CDC28-dependent phosphorylation of the C-terminal segment of Cln3 and did not affect phosphorylation of Cln2 (as was found in vivo). The reconstitution of this process with pure components provides evidence of a direct role for the chaperone in the phosphorylation of Cln3.

The regulatory particle of the Saccharomyces cerevisiae proteasome.

The proteasome is a multisubunit protease responsible for degrading proteins conjugated to ubiquitin. The 670-kDa core particle of the proteasome contains the proteolytic active sites, which face an interior chamber within the particle and are thus protected from the cytoplasm. The entry of substrates into this chamber is thought to be governed by the regulatory particle of the proteasome, which covers the presumed channels leading into the interior of the core particle. We have resolved native yeast proteasomes into two electrophoretic variants and have shown that these represent core particles capped with one or two regulatory particles. To determine the subunit composition of the regulatory particle, yeast proteasomes were purified and analyzed by gradient sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Resolution of the individual polypeptides revealed 17 distinct proteins, whose identities were determined by amino acid sequence analysis. Six of the subunits have sequence features of ATPases (Rpt1 to Rpt6). Affinity chromatography was used to purify regulatory particles from various strains, each of which expressed one of the ATPases tagged with hexahistidine. In all cases, multiple untagged ATPases copurified, indicating that the ATPases assembled together into a heteromeric complex. Of the remaining 11 subunits that we have identified (Rpn1 to Rpn3 and Rpn5 to Rpn12), 8 are encoded by previously described genes and 3 are encoded by genes not previously characterized for yeasts. One of the previously unidentified subunits exhibits limited sequence similarity with deubiquitinating enzymes. Overall, regulatory particles from yeasts and mammals are remarkably similar, suggesting that the specific mechanistic features of the proteasome have been closely conserved over the course of evolution.

p34Cdc28-mediated control of Cln3 cyclin degradation.

Cln3 cyclin of the budding yeast Saccharomyces cerevisiae is a key regulator of Start, a cell cycle event in G1 phase at which cells become committed to division. The time of Start is sensitive to Cln3 levels, which in turn depend on the balance between synthesis and rapid degradation. Here we report that the breakdown of Cln3 is ubiquitin dependent and involves the ubiquitin-conjugating enzyme Cdc34 (Ubc3). The C-terminal tail of Cln3 functions as a transferable signal for degradation. Sequences important for Cln3 degradation are spread throughout the tail and consist largely of PEST elements, which have been previously suggested to target certain proteins for rapid turnover. The Cln3 tail also appears to contain multiple phosphorylation sites, and both phosphorylation and degradation of Cln3 are deficient in a cdc28ts mutant at the nonpermissive temperature. A point mutation at Ser-468, which lies within a Cdc28 kinase consensus site, causes approximately fivefold stabilization of a Cln3-beta-galactosidase fusion protein that contains a portion of the Cln3 tail and strongly reduces the phosphorylation of this protein. These data indicate that the degradation of Cln3 involves CDC28-dependent phosphorylation events.

Inhibition of proteolysis and cell cycle progression in a multiubiquitination-deficient yeast mutant.

The degradation of many proteins requires their prior attachment to ubiquitin. Proteolytic substrates are characteristically multiubiquitinated through the formation of ubiquitin-ubiquitin linkages. Lys-48 of ubiquitin can serve as a linkage site in the formation of such chains and is required for the degradation of some substrates of this pathway in vitro. We have characterized the recessive and dominant effects of a Lys-48-to-Arg mutant of ubiquitin (UbK48R) in Saccharomyces cerevisiae. Although UbK48R is expected to terminate the growth of Lys-48 multiubiquitin chains and thus to exert a dominant negative effect on protein turnover, overproduction of UbK48R in wild-type cells results in only a weak inhibition of protein turnover, apparently because the mutant ubiquitin can be removed from multiubiquitin chains. Surprisingly, expression of UbK48R complements several phenotypes of polyubiquitin gene (UB14) deletion mutants. However, UbK48R cannot serve as a sole source of ubiquitin in S. cerevisiae, as evidenced by its inability to rescue the growth of ubi1 ubi2 ubi3 ubi4 quadruple mutants. When provided solely with UbK48R, cells undergo cell cycle arrest with a terminal phenotype characterized by replicated DNA, mitotic spindles, and two-lobed nuclei. Under these conditions, degradation of amino acid analog-containing proteins is severely inhibited. Thus, multiubiquitin chains containing Lys-48 linkages play a critical role in protein degradation in vivo.

The multiubiquitin-chain-binding protein Mcb1 is a component of the 26S proteasome in Saccharomyces cerevisiae and plays a nonessential, substrate-specific role in protein turnover.

The 26S proteasome is an essential proteolytic complex that is responsible for degrading proteins conjugated with ubiquitin. It has been proposed that the recognition of substrates by the 26S proteasome is mediated by a multiubiquitin-chain-binding protein that has previously been characterized in both plants and animals. In this study, we identified a Saccharomyces cerevisiae homolog of this protein, designated Mcb1. Mcb1 copurified with the 26S proteasome in both conventional and nickel chelate chromatography. In addition, a significant fraction of Mcb1 in cell extracts was present in a low-molecular-mass form free of the 26S complex. Recombinant Mcb1 protein bound multiubiquitin chains in vitro and, like its plant and animal counterparts, exhibited a binding preference for longer chains. Surprisingly, (delta)mcb1 deletion mutants were viable, grew at near-wild-type rates, degraded the bulk of short-lived proteins normally, and were not sensitive to UV radiation or heat stress. These data indicate that Mcb1 is not an essential component of the ubiquitin-proteasome pathway in S.cerevisiae. However, the (delta)mcb1 mutant exhibited a modest sensitivity to amino acid analogs and had increased steady-state levels of ubiquitin-protein conjugates. Whereas the N-end rule substrate, Arg-beta-galactosidase, was degraded at the wild-type rate in the (delta)mcb1 strain, the ubiquitin fusion degradation pathway substrate, ubiquitin-Pro-beta-galactosidase, was markedly stabilized. Collectively, these data suggest that Mcb1 is not the sole factor involved in ubiquitin recognition by the 26S proteasome and that Mcb1 may interact with only a subset of ubiquitinated substrates.

Structure and mechanism of ATP-dependent proteases.

A general paradigm for energy-dependent proteases is emerging: ATP may be used to unfold the substrate and translocate it through a narrow channel within the enzyme into a central proteolytic chamber. Different members of the family present intriguing elaborations on this model.

The effects of single-dose atenolol, labetalol, and propranolol on cardiac and vascular function.

The pharmacodynamic effects of single oral doses of atenolol (100 mg), labetalol (300 mg), and propranolol (80 mg) were compared with those of placebo in a randomized, double-blind, Latin square design in 12 patients with hypertension. Atenolol and propranolol both significantly reduced cardiac output (-0.55 vs. -0.31 L/min) and heart rate (-8.0 vs. -6.6 bpm), whereas labetalol had no effect on either parameter (-0.08 L/min; + 1.0 bpm). Labetalol significantly reduced vascular resistance (-339 dynes X cm/sec5), but atenolol and propranolol did not (147 vs. 62 dynes X cm/sec5). Only labetalol significantly reduced the systolic (-15.3 mm Hg), diastolic (-11.5 mm Hg), and mean blood pressures (-12.8 mm Hg). Atenolol significantly reduced only diastolic blood pressure (-5.20 mm Hg), whereas propranolol failed to lower these parameters significantly. These data indicate that the hemodynamic profile of labetalol differs from that of selective and nonselective beta-blockers. Labetalol lowered blood pressure primarily by reducing vascular resistance, whereas reductions in heart rate and cardiac output were the predominant effects of atenolol and propranolol.

The pharmacodynamic and pharmacokinetic interaction between single doses of flecainide acetate and verapamil: effects on cardiac function and drug clearance.

Flecainide and verapamil are antiarrhythmic agents that may be used in combination. We have examined their pharmacodynamic interaction by M-mode echocardiography and electrocardiography in eight normal male volunteers (24 +/- 1.8 years of age). Flecainide decreased the left ventricular ejection fraction (LVEF) (-4.4 +/- 1.2%, p less than 0.008), but verapamil did not. Neither drug affected cardiac output or vascular resistance. Both drugs increased the PR interval (12 +/- 4 msec, p less than 0.01 for flecainide; 12 +/- 5, p less than 0.04 for verapamil). Flecainide, but not verapamil, increased the QTc interval (23 +/- 8 msec, p less than 0.02). Both drugs also increased the systolic time interval ratio (PEPc/LVETc) (0.074 +/- 0.012, p less than 0.0004 for flecainide; 0.029 +/- 0.008, p less than 0.007 for verapamil). The combination of flecainide and verapamil had additive effects on myocardial contractility and on atrioventricular conduction. Verapamil slightly decreased the plasma clearance of flecainide (7.78 +/- 0.60 ml/kg/min for flecainide alone, 7.34 +/- 0.48 ml/kg/min for flecainide and verapamil together, p less than 0.05). On the other hand, flecainide had no effect on the plasma clearance of verapamil, which suggests that there was little interaction between the two drugs on their pharmacokinetic parameters.

The effects of several weeks of ethanol consumption on ethanol kinetics in normal men and women.

We examined in normal men and women the effects of chronic ethanol consumption and the coadministration of cimetidine and ranitidine on the kinetics of ethanol. We found that the consumption of 45 gm ethanol per day for 3 weeks increased the apparent volume of distribution of ethanol in men from 732 to 884 ml/kg (P less than 0.01) but had no such effect in women (697 ml/kg before ethanol and 746 ml/kg after chronic ethanol consumption). This combined therapy had no effect on the rate of ethanol disappearance in either sex. In men the rate of disappearance was 165 mg/L/hr before and 168 mg/L/hr after chronic consumption, while in women the respective values were 209 and 203 mg/L/hr. The addition of either cimetidine or ranitidine had no effect on either parameter compared with values observed on day 22 of the study. In view of the known inhibitory effects of cimetidine on cytochrome P-450-dependent enzymes, our data suggest that this enzyme system does not metabolize a significant fraction of ingested ethanol in subjects who have consumed moderate doses of alcohol for several weeks.