Search for CP Violation in Neutrino and Antineutrino Oscillations by the T2K Experiment with 2.2×10

The T2K experiment measures muon neutrino disappearance and electron neutrino appearance in accelerator-produced neutrino and antineutrino beams. With an exposure of 14.7(7.6)×10^{20} protons on target in the neutrino (antineutrino) mode, 89 ν_{e} candidates and seven anti-ν_{e} candidates are observed, while 67.5 and 9.0 are expected for δ_{CP}=0 and normal mass ordering. The obtained 2σ confidence interval for the CP-violating phase, δ_{CP}, does not include the CP-conserving cases (δ_{CP}=0, π). The best-fit values of other parameters are sin^{2}θ_{23}=0.526_{-0.036}^{+0.032} and Δm_{32}^{2}=2.463_{-0.070}^{+0.071}×10^{-3} eV^{2}/c^{4}.

Combined Analysis of Neutrino and Antineutrino Oscillations at T2K.

T2K reports its first results in the search for CP violation in neutrino oscillations using appearance and disappearance channels for neutrino- and antineutrino-mode beams. The data include all runs from January 2010 to May 2016 and comprise 7.482×10^{20} protons on target in neutrino mode, which yielded in the far detector 32 e-like and 135 µ-like events, and 7.471×10^{20} protons on target in antineutrino mode, which yielded 4 e-like and 66 µ-like events. Reactor measurements of sin^{2}2θ_{13} have been used as an additional constraint. The one-dimensional confidence interval at 90% for the phase δ_{CP} spans the range (-3.13, -0.39) for normal mass ordering. The CP conservation hypothesis (δ_{CP}=0, π) is excluded at 90% C.L.

Enhanced potency of human calcitonin when fibrillation is avoided.

The peptide hormone calcitonin (CT) is a potent drug for the therapy of different bone diseases. Salmon CT (sCT) is reported to be more active than human CT (hCT). Human CT, but not sCT, has a strong tendency to aggregate and fibrillate in aqueous solutions. Recent investigations of the fibrillation mechanisms contributed to the development of hCT solutions in which fibrillation is inhibited. Taking into consideration these new findings, we tested the relative activities of hCT handled so as to avoid aggregation/fibrillation, sCT handled in exactly the same way, and hCT handled carefully but without regard to possible fibrillation (denoted R-hCT). The effect of the CTs on bone resorption by isolated osteoclasts was measured. This assay measures the activity of interest (bone resorption) by the cell (the osteoclast) at which therapy is directed. The concentration that inhibits 50% of resorption (EC50) for hCT is 10(-5)-10(-4) pg/mL, compared with 10(-2)-1 pg/mL for R-hCT and 10(-3)-10(-1) pg/mL for sCT. The results show that when aggregation and fibrillation are avoided, hCT at the EC50 is 2-4 orders of magnitude more active than R-hCT. Thus, earlier reports of lower potency of hCT compared with sCT may have been based on inadvertent use of partially aggregated/fibrillated samples of hCT. This finding may have implications for the dose and dosage forms advised for human therapy.

Ipriflavone directly inhibits osteoclastic activity.

Ipriflavone, an isoflavone derivative, is a new drug used in an attempt to decrease bone loss in osteoporosis. Experimental studies have shown that this compound acts by inhibiting osteoclastic bone resorption both in vivo and in vitro, but the mechanism of its inhibitory action on resorbing cells remains unclear. Using bone resorption assays, video image analysis together with measurements of intracellular free calcium in isolated osteoclasts, we show here that IP directly inhibits osteoclastic activity by the modulation of intracellular free calcium.

The structure and mechanism of formation of human calcitonin fibrils.

Turbidity measurements of the kinetics of human calcitonin (hCT) fibrillation showed a linear dependence of the logarithm of fibrillation time (the time the sample is not fibrillated) and the logarithm of hCT concentration. This ln/ln plot linearity and electron microscope observations of fibrils indicate that the fibrillation process can be explained by the double nucleation mechanism that was proposed for the gelation of sickle cell hemoglobin (Ferrone, F. A., Hofrichter, J., Sunshine, H. R., and Eaton, W. A. (1980) Biophys. J. 32, 361-380). Circular dichroism, fluorescence, and infrared spectroscopy studies of fibrils showed that hCT molecules have alpha-helical and beta-sheet secondary structure components. A model for the structure of hCT molecules in fibrils is proposed.

Formation of cage-like particles by poly(amino acid)-based block copolymers in aqueous solution.

When dissolved in N,N-dimethylformamide and then dialyzed against phosphate-buffered saline, A-B-A block copolymers composed of poly [N5-(2-hydroxyethyl)-L-glutamine]-block-poly(gamma-benzyl-L-glutamate)- block-poly [N5-(2-hydroxyethyl)-L-glutamine] form particles. The particles are cage-like structures with average diameters of 300 nm (average polydispersity, 0.3-0.5). They are stable in aqueous solution at 4 degrees C for up to 3 weeks, at which time flocculation becomes apparent. Negative staining and freeze-fracture electron microscopy suggest that cage-like particles are formed by selective association of segregated micelle populations. A model of particle formation is presented in which B blocks form micelles in dimethylformamide. On dialysis against an aqueous solution, the extended A blocks then associate intermolecularly to form rod-shaped micelles, which connect the B block micelles. The result is a meshed cage-like particle. The implications of these observations on the aggregation behavior of polymeric surfactants in dilute solution are discussed.

Specific interaction of CD4-bearing liposomes with HIV-infected cells.

The CD4 molecule was reconstituted into the bilayers of large liposomes. Fluorescence microscopy and electron microscopy showed that these liposomes interact with HIV-infected H9-HT cells, delivering their contents to the cell interior. Liposomes bearing CD4 did not interact in this way with noninfected H9-HT cells nor did liposomes without CD4 interact significantly with HIV-infected cells. From electron micrographs, it appeared that HIV binds to liposomes bearing CD4; no attachment of virions to liposomes without CD4 was observed.

Dextran protection of erythrocytes from low-pH-induced hemolysis.

Low-pH-induced hemolysis of erythrocytes is inhibited by dextrans. The protective effect was observed with dextrans larger than 40 kDa. Electron microscopy showed dextrans of 150 kDa in a tight association with the erythrocyte membrane. These results indicate that dextrans stop the low-pH-induced hemolysis by interacting with the acid-induced defects in the erythrocyte membrane [(1989) Biochim. Biophys. Acta, in press].

Low-pH association of proteins with the membranes of intact red blood cells. I. Exogenous glycophorin and the CD4 molecule.

Glycophorin and CD4 proteins are tightly associated with intact human erythrocyte membranes after a short-time incubation at low pH (1-2 min, pH lower than 5, 37 degrees C). Flow cytometry and epifluorescence microscope observations showed that after incubation of red cells with fluorescein isothiocyanate (FITC) labeled glycophorin at pH values lower than 5, the erythrocyte membrane and subsequently formed ghost membranes were fluorescent. Unlabeled glycophorin was reacted with mouse erythrocytes using the same low-pH conditions. Flow cytometry and fluorescence microscopy showed that anti-glycophorin monoclonal antibodies were able to recognize the epitopes of glycophorin associated with the mouse erythrocytes. Kinetic experiments showed that the interaction of FITC-glycophorin with red cell membranes can be monitored by a decrease in the fluorescence intensity. Erythrocyte associated glycophorin was not removed from the membranes after 24 h incubation in human plasma (in vitro, 39 degrees C). A glycoprotein extract containing CD4 was isolated from a T4-lymphoma cell line (CEM). This protein extract was incubated with erythrocytes using the same low-pH conditions. Fluorescently labeled monoclonal antibodies against CD4 stained the red cells after association of CD4 with the membranes. Electron microscopy showed 10 nm immunoglobulin G-coated gold beads associated with CD4-bearing erythrocyte membranes after incubation with anti-CD4 antibodies and then with the gold beads. The potential use of the CD4-erythrocyte complex as a therapeutical agent against acquired immune deficiency syndrome (AIDS) is suggested.

Low-pH association of proteins with the membranes of intact red blood cells. II. Studies of the mechanism.

The low-pH interaction of proteins with erythrocyte membranes has been found to be correlated with pH-induced changes in the erythrocyte membrane. Using a 90 degree lightscattering method it was shown that red blood cell hemolysis was slow between pH 5.8 and 5 (t1/2 above 1 h) but became fast at and below pH 4.7 (t1/2 less than 20 min). At pH 4.7, the presence of glycophorin in the incubation medium inhibited the hemolysis of erythrocytes and this protective effect was found to be dependent on the glycophorin concentration. Electron microscope experiments showed the presence of membrane defects after 10 s incubation at pH 4.6 in the absence of glycophorin in the incubation medium. These defects could further develop into openings with average widths of 14 nm after 1.5 min incubation under the acidic conditions. Fluorescence and flow cytometry studies showed that at pH 4.7, but not at pH 7.4, glycophorin tightly associates with phosphatidylcholine liposomes, and that liposome associated glycophorin molecules are recognized by anti-glycophorin monoclonal antibodies.

Liposomes as carriers of DNA.

A recombinant plasmid encoding for rat preproinsulin I was encapsulated in large liposomes and injected intravenously into rats. Glycemia and blood, splenic and hepatic insulin were assayed from 6 h after inoculation. Control animals received (1) empty liposomes, (2) liposomes carrying the E. coli pBR322 plasmid, (3) the free rat insulin I gene, or (4) no injection. All controls showed unchanged glucose and insulin levels. Six hours after inoculation, the treated rats had 72 +/- 6 mg glucose per 100 ml of blood, compared with 107 +/- 2 mg for controls. Radioimmunoassay of blood insulin gave 61 +/- 8 microU/ml (43 +/- 5 microU/Ml for controls). Spleen and liver values were 242 +/- 20 and 205 +/- 22 microU/g of tissue, respectively (112 +/- 20 and 87 +/- 15 microU/g in controls). The kinetics and extent of uptake of liposomes by spleen and liver were studied by external gamma-camera imaging after injection of 111In-labeled liposomes. The results paralleled insulin synthesis in the two organs. The insulin gene was localized in liver cells after the injection of liposomes containing the plasmid encoding insulin. Livers were processed 4 h after inoculation for isolation of hepatocytes, Kupffer cells, and endothelial cells. DNA was purified and exogenous DNA detected by Southern blotting. Kupffer cells were the primary targets for gene incorporation with liposomes consisting of phospholipids and cholesterol. Targeting of liposomes to other liver cells was attempted by including lactosylceramide in the liposomes. This increased the amount of exogenous gene in hepatocytes and particularly in endothelial cells. Detailed electron microscopy and biochemical studies were performed in order to follow the liposome-encapsulated DNA from the moment of i.v. injection to its entering the nucleus of different liver cells. Interactions of liposomes taken up in vivo by the liver cells with subcellular organelles were studied as well. The mechanism of DNA transport by liposomes in vivo and its potential are discussed.

On the use of Percoll as a marker for liposomes in electron microscopy of mouse spleen tissue.

Liposomes encapsulating Percoll were prepared by reverse phase evaporation and characterized by electron microscopy (EM). Encapsulated Percoll was contained between lipid bilayers and in the lumen of the liposomes. The largest population of liposomes had diameters between 110 and 140 nm (22.6 per cent of total vesicle population). Percoll proved to be a good marker for liposomes in spleen tissue. It is inherently electron dense, has dimensions which are uniform and not easily confused with subcellular organelles, and can be encapsulated and visualized by EM examination of tissue by routine procedures. Electron microscope examination of spleen tissue excised 20 min after intravenous injection of liposomes encapsulating Percoll showed the presence of both liposome-encapsulated Percoll and free Percoll in phagolysosomes, suggesting that liposomes had been phagocytosed by macrophages in a way similar to circulating blood cells and that enzymatic digestion had already begun. On the other hand, intravenously injected free Percoll did not localize in phagolysosomes, but its fate appeared similar to that of intravenously injected colloidal carbon, being phagocytosed by what appeared to be macrophages and platelets.

Interaction of intravenously injected liposomes with mouse liver mitochondria. A fluorescence and electron microscopy study.

Megamitochondria, resulting from cuprizone feeding of Swiss ICR mice, were fluorescent in hepatocytes after the intravenous injection to mice of a liposome-encapsulated acridine orange-DNA complex (AO-DNA). Flow cytofluorimetric analysis of isolated megamitochondria showed that the proportion of liposome-encapsulated AO-DNA which localized in megamitochondria increased from 0.02% of the dose injected per liver cell at 3 min after injection to an average of 0.34% at 1 h after injection. Megamitochondria showed negligible fluorescence by fluorescence activated cell sorting (FACS) analysis when free AO-DNA was intravenously injected. Transmission electron micrographs of mouse liver tissue after intravenous injection of liposomes encapsulating iron dextran showed an association of the liposomes with megamitochondria which appeared identical to liposome association with normal mitochondria. These results support and extend our earlier observation that a fraction of the liposomes injected intravenously into mice associate with mitochondria in the liver, and possibly deliver their aqueous contents there.

Intracellular fate of liposome-encapsulated DNA in mouse liver. Analysis using electron microscope autoradiography and subcellular fractionation.

Transient expression of liposome-encapsulated DNA in liver after intravenous injection to rats and mice has raised questions concerning the intracellular fate of this DNA. Electron microscope autoradiography shows that at 10 min after injection the highest concentration of liposomal DNA which is taken up by the liver is associated with lysosomes and vesicles. The proportion of DNA associated with the mitochondria steadily increases for 1 h after injection, up to 48% of the exogenous DNA found in the tissue. Part of this DNA follows the subcellular fractionation profile of the mitochondrial matrix marker, malate dehydrogenase. In contrast, 14% of the liposomal DNA taken up by the liver is found in the nuclei at 3 min after injection, and this percentage decreases over a period of 1 h. These results permit us to establish the distribution of liposome-encapsulated DNA among subcellular organelles in liver at different times after injection.

Liposomes injected intravenously into mice associate with liver mitochondria.

Liposomes encapsulating uranyl acetate or ferritin were injected intravenously into mice. At periods of 20 min, 1 h and 4 h post-injection, animals were killed, and livers were excised. Transmission electron micrographs of liver tissue showed association of oligolamellar liposomes with mitochondria for each time period. At 1 h post-injection, an average of one out of ten mitochondria was associated with liposomes. In most cases, the liposomes were clearly enclosed in a cytoplasmic vacuole. Phagocytosis by Kupffer cells as well as fusion with primary lysosomes and inclusion in secondary lysosomes was observed. No difference in intracellular fate was observed when lactosylceramide was incorporated in the liposome bilayers, suggesting that the differences observed in biochemical studies are at the level of liposome-plasma membrane interaction. When liposomes containing uranyl acetate were intravenously injected and hepatocytes were isolated by collagenase perfusion one hour later, transmission EM revealed the presence of liposomes in these cells, in cytoplasmic vacuoles in the cytoplasm and in association with mitochondria. A freeze-fracture-etching analysis of liver tissue excised 20 min after injection of liposomes encapsulating ferritin, further supported the observation that liposomes associate with mitochondria in the liver.

Electrostatic facilitation of the reaction catalyzed by the manganese-containing and the iron-containing superoxide dismutases.

Both the iron-containing and the manganese-containing superoxide dismutases from Escherichia coli show diminished activity with increasing ionic strength, indicative of electrostatic facilitation of the catalyzed reaction. Since both enzymes bear a net negative charge at the assay pH, as does the substrate, this suggests a cationic locale in the active site region. Acetylation of the enzymes inverted their response to increasing ionic strength. It thus appears that lysine residues provide the observed electrostatic facilitation. A specific inhibition by large monovalent anions was observed with the iron-containing superoxide dismutase and was taken to indicate the presence of a cationic group, within a hydrophobic crevice, at the active site.

Electrostatic interactions in the reaction mechanism of bovine erythrocyte superoxide dismutase.

The activity of the copper- and zinc-containing superoxide dismutase decreased with increasing ionic strength. Modification of lysine residues by acetylation or succinylation inverted the effect of increasing ionic strength, whereas modification of arginine with phenylglyoxal did not. These results were noted in both photochemical and pulse-radiolysis assays. It appears that interaction of O2- with the anionic enzyme is assisted by the positive charge on lysine residues, presumably those close to the active site. By the criterion of responsiveness to ionic strength, the arginine residue close to the active site does not appear to provide electrostatic facilitation to the catalytic process. Elimination of the charge on epsilon-amino groups by raising the pH suppressed activity to the same extent as did elimination of these charges by acetylation. Activity was similarly suppressed to the same extent by covalent modification or by ionization of arginine residues, indicating that the positive charge on arginine is important for the catalytic process even though its effect is not responsive to changes in the ionic strength of the solution.