Temperature-induced change of variant surface antigen expression in Paramecium involves antigen release into the culture medium with considerable delay between transcription and surface expression.

We analyzed temperature-induced changes of variant surface antigen (vsAg) expression in Paramecium primaurelia, using immuno-techniques and mRNA determinations. Upon a 23 degrees C to 33 degrees C shift, the old vsAg, type 156G, remains on the cell surface for a time, when already mRNA for the new form, 156D, is expressed. A considerable amount of 156D-specific mRNA is formed 45-48 h after the temperature shift, while 156D surface expression reaches maximal levels only after >72 h. A new aspect of these experiments is that, during this transition, the old vsAg is steadily released in high-molecular-weight form into the culture medium, as found by dot blot and Western blot analysis of concentrated culture medium. The new vsAg form is first inserted into the somatic cell membrane, before it spreads also into cilia. In the reverse transition, 33 degrees C to 23 degrees C, the adaptation on the level of transcription and surface expression is considerably faster. While we had previously shown, under steady-state conditions (constant temperature), the occurrence of a degradation pathway by endocytotic and phagocytotic ingestion of vsAg this may proceed in parallel to the steady release of old vsAg from the cell surface into the medium. Altogether these combined processes may facilitate the installation of the new vsAg type.

Fine structure in proton emission from 145Tm discovered with digital signal processing.

Fine structure in proton emission from the 3.1(3) mus activity of 145Tm was discovered by using a novel technique of digital processing of overlapping recoil implantation and decay signals. Proton transitions to the ground state of 144Er and to its first excited 2(+) state at 0.33(1) MeV with a branching ratio I(p)(2(+))=9.6+/-1.5% were observed. The structure of the 145Tm wave function and the emission process were analyzed by using particle-core vibration coupling models.

Measurement of B --> K*gamma branching fractions and charge asymmetries.

The branching fractions of the exclusive decays B0-->K(*0)gamma and B+-->K(*+)gamma are measured from a sample of (22.74+/-0.36)x10(6) BB decays collected with the BABAR detector at the PEP-II asymmetric e(+)e(-) collider. We find B (B0-->K(*0)gamma) = [4.23+/-0.40(stat)+/-0.22(syst)]x10(-5), B(B+-->K(*+)gamma) = [3.83+/-0.62(stat)+/-0.22(syst)]x10(-5) and constrain the CP-violating charge asymmetry to be -0.170K(*)gamma)<0.082 at 90% C.L.

Measurements of the branching fractions of exclusive charmless B meson decays with eta(') or omega mesons.

We present the results of searches for B decays to charmless two-body final states containing eta(') or omega mesons, based on 20.7 fb(-1) of data collected with the BABAR detector. We find the branching fractions Beta(B(+)-->eta(')K(+)) = (70+/-8+/-5) x 10(-6), Beta(B(0)-->eta(')K(0)) = (42(+13)(-11) +/- 4) x 10(-6), and Beta(B(+)-->omega pi(+)) = (6.6(+2.1)(-1.8) +/- 0.7) x 10(-6), where the first error quoted is statistical and the second is systematic. We give measurements of four additional modes for which the 90% confidence level upper limits are Beta(B(+)-->eta(')pi(+)) < 12 x 10(-6), Beta(B(+)-->omega K(+)) < 4 x 10(-6), Beta(B(0)-->omega K(0)) < 13 x 10(-6), and Beta(B(0)-->omega pi(0)) < 3 x 10(-6).

Measurement of the B--> J/psiK*(892) decay amplitudes.

We present a measurement of the decay amplitudes in B-->J/psiK*(892) channels using 20.7 fb(-1) of data collected at the Upsilon(4S) resonance with the BABAR detector at PEP-II. We measure a P-wave fraction R(perpendicular) = (16.0 +/- 3.2 +/- 1.4)% and a longitudinal polarization fraction (59.7 +/- 2.8 +/- 2.4)%. The measurement of a relative phase that is neither 0 nor pi, phi = 2.50 +/- 0.20 +/-0.08 radians, favors a departure from the factorization hypothesis. Although the decay B-->/psiK(pi) proceeds mainly via K*(892), there is also evidence for K2*(1430) and K(pi) S-wave contributions.

Search for the decay B0-->gammagamma.

We present a limit on the branching fraction for the decay B0-->gammagamma using data collected at the Upsilon(4S) resonance with the BABAR detector at the PEP-II asymmetric energy e+e- collider. Based on the observation of one event in the signal region, out of a sample of 21.3x10(6) e+e--->Upsilon(4S)-->BB decays, we establish an upper limit on the branching fraction of B(B0-->gammagamma)<1.7x10(-6) at the 90% confidence level. This result substantially improves upon existing limits.

Measurement of J/psi production in continuum e(+)e(-) annihilations near square root of s = 10.6 GeV.

The production of J/psi mesons in continuum e(+)e(-) annihilations has been studied with the BABAR detector at energies near the Upsilon(4S) resonance. The mesons are distinguished from J/psi production in B decays through their center-of-mass momentum and energy. We measure the cross section e(+)e(-)-->J/psi X to be 2.52+/-0.21+/-0.21 pb. We set a 90% C.L. upper limit on the branching fraction for direct Upsilon(4S)-->J/psi X decays at 4.7 x 10(-4).

Measurement of the B(0) and B(+) meson lifetimes with fully reconstructed hadronic final states.

The B(0) and B(+) meson lifetimes have been measured in e(+)e(-) annihilation data collected in 1999 and 2000 with the BABAR detector at center-of-mass energies near the Upsilon(4S) resonance. Events are selected in which one B meson is fully reconstructed in a hadronic final state while the second B meson is reconstructed inclusively. A combined fit to the B(0) and the B(+) decay time difference distributions yields tau(B(0)) = 1.546+/-0.032(stat)+/-0.022(syst) ps, tau(B(+)) = 1.673+/-0.032(stat)+/-0.023(syst) ps, and tau(B(+))/tau(B(0)) = 1.082+/-0.026(stat)+/-0.012(syst).

Measurement of the decays B--> phiK and B--> phiK*.

We have observed the decays B--> phiK and phiK(*) in a sample of over 45 million B mesons collected with the BABAR detector at the PEP-II collider. The measured branching fractions are B(B+--> phiK+) = (7.7(+1.6)(-1.4)+/-0.8)x10(-6), B(B0--> phiK0) = (8.1(+3.1)(-2.5)+/-0.8)x10(-6), B(B+--> phiK(*+)) = (9.7(+4.2)(-3.4)+/-1.7)x10(-6), and B(B0--> phiK(*0)) = (8.7(+2.5)(-2.1)+/-1.1)x10(-6). We also report the upper limit B(B+--> phipi(+))<1.4x10(-6) ( 90% C.L.).

Measurement of branching fractions and search for CP-violating charge asymmetries in charmless two-body B decays into pions and kaons.

We present measurements, based on a sample of approximately 23x10(6) BB pairs, of the branching fractions and a search for CP-violating charge asymmetries in charmless hadronic decays of B mesons into two-body final states of kaons and pions. We find the branching fractions B(B0-->pi(+)pi(-)) = (4.1+/-1.0+/-0.7)x10(-6), B(B0-->K+pi(-)) = (16.7+/-1.6+/-1.3)x10(-6), B(B+-->K+pi(0)) = (10.8(+2.1)(-1.9)+/-1.0)x10(-6), B(B+-->K0pi(+)) = (18.2(+3.3)(-3.0)+/-2.0)x10(-6), B(B0-->K0pi(0)) = (8.2(+3.1)(-2.7)+/-1.2)x10(-6). We also report 90% confidence level upper limits for B meson decays to the pi(+)pi(0), K+K-, and K0K+ final states. In addition, charge asymmetries have been found to be consistent with zero, where the statistical precision is in the range of +/-0.10 to +/-0.18, depending on the decay mode.

Observation of CP violation in the B(0) meson system.

We present an updated measurement of time-dependent CP-violating asymmetries in neutral B decays with the BABAR detector at the PEP-II asymmetric B Factory at SLAC. This result uses an additional sample of Upsilon(4S) decays collected in 2001, bringing the data available to 32 x 10(6) BB macro pairs. We select events in which one neutral B meson is fully reconstructed in a final state containing charmonium and the flavor of the other neutral B meson is determined from its decay products. The amplitude of the CP-violating asymmetry, which in the standard model is proportional to sin2 beta, is derived from the decay time distributions in such events. The result sin2 beta = 0.59+/-0.14(stat)+/-0.05(syst) establishes CP violation in the B(0) meson system. We also determine absolute value of lambda = 0.93+/-0.09(stat)+/-0.03(syst), consistent with no direct CP violation.

Digital pulse processing: new possibilities in nuclear spectroscopy

Digital pulse processing is a signal processing technique in which detector (preamplifier output) signals are directly digitized and processed to extract quantities of interest. This approach has several significant advantages compared to traditional analog signal shaping. First, analyses can be developed which take pulse-by-pulse differences into account, as in making ballistic deficit compensations. Second, transient induced charge signals, which deposit no net charge on an electrode, can be analyzed to give, for example, information on the position of interaction within the detector. Third, deadtimes from transient overload signals are greatly reduced, from tens of micros to hundreds of ns. Fourth, signals are easily captured, so that more complex analyses can be postponed until the source event has been deemed "interesting". Fifth, signal capture and processing may easily be based on coincidence criteria between different detectors or different parts of the same detector. XIAs recently introduced CAMAC module, the DGF-4C, provides many of these features for four input channels, including two levels of digital processing and a FIFO for signal capture for each signal channel. The first level of digital processing is "immediate", taking place in a gate array at the 40 MHz digitization rate, and implements pulse detection, pileup inspection, trapezoidal energy filtering, and control of an external 25.6 micros long FIFO. The second level of digital processing is provided by a digital signal processor (DSP), where more complex algorithms can be implemented. To illustrate digital pulse processing's possibilities, we describe the application of the DGF-4C to a series of experiments. The first, for which the DGF was originally developed, involves locating gamma-ray interaction sites within large segmented Ge detectors. The goal of this work is to attain spatial resolutions of order 2 mm sigma within 70 mm x 90 mm detectors. We show how pulse shape analysis allows ballistic deficit to be significantly reduced in these detectors. A second experiment involves studying exotic nuclei by observing their 1 MeV direct proton decays following implantation in a Si crossed stripe detector at 35 MeV. Whereas the implantation paralyzes analog electronics for almost 10 micros, the DGF allows the study of decay times as short as 1 micros. Initial energy and time resolution results are presented. Finally, we show how the DGF's precise timing and coincidence capabilities lead to significant experimental simplifications in dealing with phoswich detectors, low background counting work, and trace Pb detection by coincident photon detection.

Quantitative immunogold localization of protein phosphatase 2B (calcineurin) in Paramecium cells.

For immunogold EM labeling analysis, we fixed Paramecium cells in 4% formaldehyde and 0.125% glutaraldehyde, followed by low-temperature embedding in unicryl and UV polymerization. We first quantified some obvious but thus far neglected side effects of section staining on immunogold labeling, using mono- or polyclonal antibodies (Abs) against defined secretory and cell surface components, followed by F(ab)(2)- or protein A-gold conjugates. Use of alkaline lead staining resulted in considerable rearrangement and loss of label unless sections were postfixed by glutaraldehyde after gold labeling. This artifact is specific for section staining with lead. It can be avoided by staining sections with aqueous uranyl acetate only to achieve high-resolution immunogold localization of a protein phosphatase on unicryl sections. In general, phosphatases are assumed to be closely, although loosely, associated with their targets. Because the occurrence of protein phosphatase 2B (calcineurin) in Paramecium has been previously established by biochemical and immunological work, as well as by molecular biology, we have used Abs against mammalian CaN or its subunits, CaN-A and CaN-B, for antigen mapping in these cells by quantitative immunogold labeling analysis. Using ABs against whole CaN, four structures are selectively labeled (with slightly decreasing intensity), i.e., infraciliary lattice (centrin-containing contractile cortical filament network), parasomal sacs (coated pits), and outlines of alveolar sacs (subplasmalemmal calcium stores, tightly attached to the cell membrane), as well as rims of chromatin-containing nuclear domains. In other subcellular regions, gold granules reached densities three to four times above background outside the cell but there was no selective enrichment, e.g., in cilia, ciliary basal bodies, cytosol, mitochondria, trichocysts (dense-core secretory organelles), and non-chromatin nuclear domains. Their labeling density was 4- to 8.5-fold (average 6.5-fold) less than that on selectively labeled structures. Labeling tendency was about the same with Abs against either subunit. Our findings may facilitate the examination of molecular targets contained in the selectively labeled structures. (J Histochem Cytochem 48:1269-1281, 2000)

Hydrophobic and hydrophilic radio-iodination, crosslinking, and differential extraction of cell surface proteins in Paramecium tetraurelia cells.

We combined widely different biochemical methods to analyze proteins of the cell surface of P. tetraurelia since so far one can isolate only a subfraction of cell membrane vesicles enriched in the GPI-anchored surface antigens ("immoblization" or "i-AGs"). We also found that i-AGs may undergo partial degradation by endogenous proteases. Genuine intrinsic membrane proteins were recognized particularly with lipophilic 5-[(125)I]-iodonaphthalene-1-azide (INA) labeling which reportedly "sees" integral proteins and cytoplasmic cell membrane-associated proteins. With INA (+DTT), bands of

Microdomain arrangement of the SERCA-type Ca2+ pump (Ca2+-ATPase) in subplasmalemmal calcium stores of paramecium cells.

We localized SERCA pumps to the inner region of alveolar sac membranes, facing the cell interior, by combining ultrastructural and biochemical methods. Immunogold labeling largely predominated in the inner alveolar sac region which displayed aggregates of intramembrane particles (IMPs). On image analysis, these represented oligomeric arrangements of approximately 8-nm large IMP subunits, suggesting formation of SERCA aggregates (as known from sarcoplasmic reticulum). We found not only monomers of typical molecular size ( approximately 106 kD) but also oligomeric forms on Western blots (using anti-SERCA antibodies, also against endogenous SERCA from alveolar sacs) and on electrophoresis gelautoradiographs of 32P-labeled phosphoenzyme intermediates. Selective enrichment of SERCA-pump molecules in the inner alveolar sac membrane region may eliminate Ca2+ after centripetal spread observed during exocytosis activation, while the plasmalemmal Ca2+ pump may maintain or reestablish [Ca2+] in the narrow subplasmalemmal space between the outer alveolar sac membrane region and the cell membrane. We show for the first time the microzonal arrangement of SERCA molecules in a Ca2+ store of a secretory system, an intensely discussed issue in stimulus-secretion coupling research.

Immunolabeling analysis of biosynthetic and degradative pathways of cell surface components (glycocalyx) in Paramecium cells.

Biosynthetic and degradative pathways of glycocalyx components are largely unknown in Paramecium and in some related parasitic protozoa. We isolated cell surface (glyco-)proteins, i.e., surface antigens (SAg) and used them in the native (nSAg) or denatured (dSAg) state to produce antibodies (AB) for immunolocalization by confocal imaging and by quantitative immunogold EM-labeling of ultrathin sections or of freeze-fracture replicas. Antibodies against nSAg or dSAg, respectively, yield different labeling densities over individual structures, thus indicating biosynthetic or degradative pathways, respectively. We derive the following biosynthetic way: ER --> Golgi apparatus --> non-regulated/non-dense core vesicle transport --> diffusional spread over non-ciliary (somatic) and ciliary cell membrane. For degradation we show the following pathways: Concentration of nSAg in the cytostome --> nascent digestive vacuole --> mature vacuoles --> release of dSAg at cytoproct, with partial retrieval by "discoidal vesicles". A second internalization pathway proceeds via coated pits ("parasomal sacs") --> early endosomes ("terminal cisternae") --> digestive vacuoles. Dense packing of SAg in the glycocalyx may drive them into the endo-/phagocytic pathway. Still more intriguing is the site of nSAg integration into the cell membrane by unstimulated exocytosis. We consider unconspicuous clear vesicles relevant for nSAg export, probably via sites which most of the time are occupied by coated pits. This could compensate for membrane retrieval by coated pits, while scarcity of smooth profiles at these sites may be explained by the much longer time period required for coated pit formation as compared to exocytosis.

Immunolocalization of the exocytosis-sensitive phosphoprotein, PP63/parafusin, in Paramecium cells using antibodies against recombinant protein.

We have localized a structure-bound fraction of the exocytosis-sensitive phosphoprotein, PP63/parafusin (PP63/pf), in Paramecium cells by widely different methods. We combined cell fractionation, western blots, as well as light and electron microscopy (pre- and post-embedding immunolabeling), applying antibodies against the recombinant protein. PP63/pf is considerably enriched in certain cortical structures, notably the outlines of regular surface fields (kinetids), docking sites of secretory organelles (trichocysts) and the membranes of subplasmalemmal Ca2+-stores (alveolar sacs). From our localization studies we tentatively derive several potential functions for PP63/pf, including cell surface structuring, assembly of exocytosis sites, and/or Ca2+ homeostasis.

Functional characterization and localization of protein phosphatase type 2C from Paramecium.

We cloned a protein phosphatase 2C gene from Paramecium (PtPP2C), which codes for one of the smallest PP2C isoforms (Klumpp, S., Hanke, C., Donella-Deana, A., Beyer, A., Kellner, R., Pinna, L. A., and Schultz, J. E. (1994) J. Biol. Chem. 269, 32774-32780). After mutation of 9 ciliate Q codons (TAA) to CAA PtPP2C was expressed as an active protein in Escherichia coli. The catalytic core region contains 284 amino acids as defined by C- and N-terminal deletions. The C terminus from amino acid 200-300 of PP2C isoforms has only about 20% similarity. To demonstrate that the carboxy end is in fact needed for activity, we generated an enzymatically active PtPP2C containing a C-terminally located tobacco etch virus-protease site. Upon proteolytic truncation enzyme activity was lost, i.e. the C terminus of PP2C is indispensable for enzyme activity. During these experiments isoleucine 214 was fortuitously identified to be essential for PP2C catalysis. Mutation of the hydrophobic amino acid to glycine in the ciliate or bovine isoforms resulted in inactive protein. Because Ile214 is in a loop region without defined secondary structure, our data clearly go beyond the x-ray structure. The functional equivalence of the 180 amino acid long C terminus from the bovine PP2C with the 100 amino acid long carboxy end of the PtPP2C was demonstrated by producing an active chimera, i.e. the PP2C from Paramecium has no obvious regions which may be specifically involved in subcellular localization or substrate recognition. Using antibodies against recombinant PtPP2C we localized the enzyme by immunogold labeling in the cytosol and nucleus and very distinctly on the ciliary microtubule/dynein complex. The data suggest a role for PtPP2C in the regulation of dyneins, i.e. in cellular cargo transport and ciliary motility.

Immunolocalization of protein phosphatase type 1 in Paramecium cells using antibodies against recombinant protein and peptides.

We localized protein phosphatase Type 1 (PP1) in Paramecium cells using antibodies (specified on Western blots) against recombinant protein, amino- or carboxy-terminal peptides, or peptide segments containing both terminals and an intermediate segment. Cell fractionation and ELISA revealed high PP1 concentrations in cilia, corresponding to observations by immunofluorescence and immunogold labeling analyses. We compared ELISA results obtained with MnCl2- or detergent-mediated deciliation and immunolocalizations obtained with digitonin and saponin- or detergent-mediated permeabilization. We observed that detergents at too high concentrations can displace the antigen from its original position. Quantitative evaluation of immunogold labeling revealed a predominant localization of PP1 in cilia, notably in the narrow space between the membrane and the outer microtubule doublets, as ascertained by immunogold labeling of Lowicryl sections obtained after rapid freezing and freeze-substitution. This localization to the periphery of cilia is compatible with previous suggestions of PP1 involvement in ciliary beat regulation, notably of cilia on the free cell surface. Immunolabeling occurs along the entire length of surface cilia. Despite much higher PP1 concentrations in cilia, ELISA values for absolute PP1 content were considerably higher in deciliated cells. This may indicate still other functional aspects of PP1. Along these lines, we also discuss the differences observed when immunochemical and enzymatic data are compared.