Measurement of Electron-Neutrino Charged-Current Cross Sections on

Using an 185-kg NaI[Tl] array, COHERENT has measured the inclusive electron-neutrino charged-current cross section on ^{127}I with pion decay-at-rest neutrinos produced by the Spallation Neutron Source at Oak Ridge National Laboratory. Iodine is one the heaviest targets for which low-energy (≤50 MeV) inelastic neutrino-nucleus processes have been measured, and this is the first measurement of its inclusive cross section. After a five-year detector exposure, COHERENT reports a flux-averaged cross section for electron neutrinos of 9.2_{-1.8}^{+2.1}×10^{-40} cm^{2}. This corresponds to a value that is ∼41% lower than predicted using the MARLEY event generator with a measured Gamow-Teller strength distribution. In addition, the observed visible spectrum from charged-current scattering on ^{127}I has been measured between 10 and 55 MeV, and the exclusive zero-neutron and one-or-more-neutron emission cross sections are measured to be 5.2_{-3.1}^{+3.4}×10^{-40} and 2.2_{-0.5}^{+0.4}×10^{-40} cm^{2}, respectively.

Two distinct antigen-specific suppressor factors induced by the oral administration of antigen.

The feeding of sheep erythrocytes (SRBC) to mice leads to the production of two distinct T cell-derived suppressor factors by spleen cells. Each has been characterized for specificity, genetic restrictions, and cellular interactions. Fraction I has a 60,000-75,000 mol wt, is specific for antigen, and is suppressive of primary in vitro anti-SRBC responses at all times. It is not restricted by major histocompabitility complex (MHC)- or Igh-linked genes, but it fails to suppress spleen cells derived from any strain of mouse with a B10 background. It acts on an Lyt-2+ T cell to increase suppressive activity. An antiserum has been prepared against this factor that reacts with other, unrelated T cell suppressor factors. Fraction II has an approximately 30,000-40,000 mol wt, is specific for antigen, and has a dual effect on in vitro anti-SRBC responses. On day 3 of culture, it leads to augmentation of the response, whereas at day 5 it suppresses the response. It is not restricted by MHC genes, but it is restricted by Igh-linked genets. It acts by activating an Ly-1 t cell to both help and induce feedback suppression. These factors, and the antisera prepared against them, should allow more precise dissection of the molecular pathways by which immunoregulatory cells communicate with one another.

An enzyme immunoassay for the detection of all Salmonella using a combination of a myeloma protein and a hybridoma antibody.

An enzyme immunoassay was developed for the detection of salmonellae in foods and stool samples. The test is sensitive and specific, showing virtually no cross-reactivity to other enteric organisms. The sensitivity is such that less than 10(6) Salmonella organisms can be detected, and this is not influenced even by a 3 log10 overgrowth of other bacteria. This assay can be performed routinely in any laboratory, and can be automated as much as desired. It can be performed from growth in liquid or on solid media. A myeloma protein (M467) and a hybridoma antibody (6H4) were attached to a polycarbonate coated metal bead. The beads were then incubated in a heat extract of the organisms for 20 min. After thorough washing by a magnetic transfer device, the beads were then incubated in a mixture of the 2 antibodies which were labeled with horseradish peroxidase. After another thorough washing and incubation in substrate, the reactions were either read by the naked eye or by spectrophotometry. The entire process following the culture takes less than 2 h.

Enhancement of Streptococcus mutans colonization by direct bonded orthodontic appliances.

Patients undergoing orthodontic therapy were evaluated for longitudinal changes in relative S. mutans numbers in plaque at discrete sites on the tooth surface associated with direct bond appliances. There was a significant linear increase in the percentage of S. mutans in the total streptococci isolated from the last pre-bracket sample through the last bracket sample.

Teaching multiplication facts to students with learning problems.

The present study investigated the effectiveness of a constant time delay procedure in teaching multiplication facts to fifth- and sixth-grade students identified as learning disabled, behavior disordered, or educable mentally handicapped. Four students were taught oral responses to 30 multiplication facts using a constant 5-second (s) delay procedure. Data on the number of minutes of direct instruction time, as well as the number and percent of errors, trials, and sessions to criterion, were collected. Results indicated that the near-errorless learning procedure was effective in teaching multiplication facts to students placed in special education.

Local kinetic interpretation of entropy production through reversed diffusion.

The time reversal of stochastic diffusion processes is revisited with emphasis on the physical meaning of the time-reversed drift and the noise prescription in the case of multiplicative noise. The local kinematics and mechanics of free diffusion are linked to the hydrodynamic description. These properties also provide an interpretation of the Pope-Ching formula for the steady-state probability density function along with a geometric interpretation of the fluctuation-dissipation relation. Finally, the statistics of the local entropy production rate of diffusion are discussed in the light of local diffusion properties, and a stochastic differential equation for entropy production is obtained using the Girsanov theorem for reversed diffusion. The results are illustrated for the Ornstein-Uhlenbeck process.

Propagation of fluctuations in biochemical systems, II: Nonlinear chains.

We consider biochemical reaction chains and investigate how random external fluctuations, as characterised by variance and coefficient of variation, propagate down the chains. We perform such a study under the assumption that the number of molecules is high enough so that the behaviour of the concentrations of the system is well approximated by differential equations. We conclude that the variances and coefficients of variation of the fluxes will decrease as one moves down the chain and, through an example, show that there is no corresponding result for the variances of the concentrations of the chemical species. We also prove that the fluctuations of the fluxes as characterised by their time averages decrease down reaction chains. The results presented give insight into how biochemical reaction systems are buffered against external perturbations solely by their underlying graphical structure and point out the benefits of studying the out-of-equilibrium dynamics of systems.

Immunologic suppression after oral administration of antigen. III. Activation of suppressor-inducer cells in the Peyer's patches.

Mice were orally administered sheep erythrocytes (SRBC) in a regimen previously known to produce systemic tolerance to SRBC. Cellular interactions and movement from the gut-associated lymphoid tissue (GALT) to the spleen were found to occur using both in vivo and in vitro transfer systems. The cell in the GALT which initiates the suppression circuit migrates from the GALT to the spleen shortly after contacting antigen. This cell is a T suppressor-inducer (Tsi) cell which interacts with splenic lymphocytes to induce the formation of an effector T suppressor cell (Ts). The Tsi and Ts can be separated from each other by their differential sensitivities to cyclophosphamide. In addition, the Tsi can be separated from other GALT T cells by its inability to bind the lectin, peanut agglutinin. Thus, cell migration and cellular interaction among T cells must occur to result in orally induced tolerance.

Immunologic suppression after oral administration of antigen. I. Specific suppressor cells formed in rat Peyer's patches after oral administration of sheep erythrocytes and their systemic migration.

Rats given 10(10) sheep erythrocytes (SRBC) orally were found to contain specific suppressor cells to SRBC in their Peyer's patches (PP) and mesenteric lymph nodes (MLN) after 2 days of feeding. After 4 days of feeding, similar suppressor cells were found in the thymus and spleen, but they were missing in the PP or MLN. These suppressor cells effectively blocked IgM and IgG plaque-forming cell responses to SRBC in Mishell-Dutton cultures and delayed-type-hypersensitivity responses to SRBC when transferred to syngeneic recipients, but they did not affect responses to horse erythrocytes. The orally induced specific suppressor cells appeared to be T2 cells since their activity was eliminated by in vivo treatment of SRBC-fed rats with anti-rat lymphocyte serum but not by adult thymectomy. Because carrageenan partially relieved the suppression observed in culture, the actual suppressive mechanism may also involve a macrophage.

Studies on the mitogen responses of germfree allogeneic chimeras. II. Maturation of two cell types and partial restoration of responsiveness of the short-term chimeras.

Germfree allogeneic bone marrow chimeras (ABMC) were produced by the i.v. injection of approximately 10(7) bone marrow cells from germfree DBA/2 mice into lethally irradiated germfree C3H mice. In the germfree state, the short-term ABMC showed no histologic signs of graft-vs-host reactions (GVHR), yet splenic lymphocytes were unable to respond to PHA, Con A, or SRBC. Attempts to remove responsiveness by the implantation of a DBA/2 thymus under the host kidney capsule also resulted in failure. However, when the donor thymus was enclosed in a cell-impermeable chamber to eliminate a GVH reaction, responsiveness to Con A was restored. The PHA and SRBC responses were unaffected by this treatment. Daily injections of thymosin caused both an increased Con A response and increased numbers of PFC, although the PHA response was again unaffected. Thus, soluble substances from thymic tissue can be used to overcome partially the histocompatibility barrier present in the ABMC that affects at least two different functional cell populations.

The folding of nascent mitochondrial aspartate aminotransferase synthesized in a cell-free extract can be assisted by GroEL and GroES.

At 30 degrees C, the precursor to mitochondrial aspartate aminotransferase (pmAspAT) cannot fold after synthesis in rabbit reticulocyte lysate (RRL), a model for studying intracellular protein folding. However, it folds rapidly once imported into mitochondria. Guanidinium chloride denatured pmAspAT likewise cannot refold at 30 degrees C in a defined in vitro system. However, it refolds rapidly and in good yield in the presence of the intramitochondrial chaperone homologues GroEL and GroES. In this report, we demonstrate that GroEL and GroES can also facilitate the folding of nascent pmAspAT in reticulocyte lysate under conditions where it otherwise would not. When added alone, GroEL arrests the slow folding of nascent pmAspAT and inhibits import into mitochondria. These effects are significantly reversed by adding GroES. These observations suggest that added GroEL participates in an equilibrium with endogenous chaperones in the cytosol which inhibit folding and promote import competence. Native gel electrophoresis suggests that nascent pmAspAT exists in RRL as a heterogeneous population of partially folded species, some of which bind to added GroEL more readily than others. The GroEL-trapped species appear to be among the productive pmAspAT folding intermediates formed in RRL or they at least appear to equilibrate with these intermediates, since they become import competent after GroES-stimulated release from GroEL.

Homologous proteins with different affinities for groEL. The refolding of the aspartate aminotransferase isozymes at varying temperatures.

The homologous cytosolic and mitochondrial isozymes of aspartate aminotransferase (c- and mAspAT, respectively) seem to follow very different folding pathways after synthesis in rabbit reticulocyte lysate, suggesting that the nascent proteins interact differently with molecular chaperones (Mattingly, J. R., Jr., Iriarte, A., and Martinez-Carrion, M. (1993) J. Biol. Chem. 268, 26320-26327). In an attempt to discern the structural basis for this phenomenon, we have begun to study the effect of temperature on the refolding of the guanidine hydrochloride-denatured, purified proteins and their interaction with the groEL/groES molecular chaperone system from Escherichia coli. In the absence of chaperones, temperature has a critical effect on the refolding of the two isozymes, with mAspAT being more susceptible than cAspAT to diminishing refolding yields at increasing temperatures. No refolding is observed for mAspAT at physiological temperatures. The molecular chaperones groEL and groES can extend the temperature range over which the AspAT isozymes successfully refold; however, cAspAT can still refold at higher temperatures than mAspAT. In the absence of groES and MgATP, the two isozymes interact differently with groEL, groEL arrests the refolding of mAspAT throughout the temperature range of 0-45 degrees C. Adding only MgATP releases very little mAspAT from groEL; both groES and MgATP are required for significant refolding of mAspAT in the presence of groEL. On the other hand, the extent to which groEL inhibits the refolding of cAspAT depends upon the temperature of the refolding reaction, only slowing the reaction at 0 degrees C but arresting it completely at 30 degrees C. MgATP alone is sufficient to effect the release of cAspAT from groEL at any temperature examined; inclusion of groES along with MgATP has no effect on the refolding yield but does increase the refolding rate at temperatures greater than 15 degrees C. These results demonstrate that groEL can have significantly different affinities for proteins with highly homologous final tertiary and quarternary structures and suggest that dissimilarities in the primary sequence of the protein substrates may control the structure of the folding intermediates captured by groEL and/or the composition of the surfaces through which the folding proteins interact with groEL.

Structural features which control folding of homologous proteins in cell-free translation systems. The effect of a mitochondrial-targeting presequence on aspartate aminotransferase.

When the precursor to mitochondrial aspartate aminotransferase (pmAspAT) is synthesized in a rabbit reticulocyte lysate translation system (RRL), its properties are quite unlike those of the purified protein (Mattingly, J.R., Jr., Youssef, J., Iriarte, A., and Martinez-Carrion, M. (1993) J. Biol. Chem. 268, 3925-3937). These results suggest that molecular chaperones present in RRL modulate the folding of pmAspAT. To investigate the structural basis for this, we have used protease resistance to monitor the extent of folding for several related AspATs after synthesis in RRL and in wheat germ extract (WGE). In addition to pmAspAT, the following proteins were examined: the mature form of pmAspAT (delta 2-28 pmAspAT), its cytosolic counterpart (cAspAT), a chimeric protein consisting of the presequence of pmAspAT attached to the amino terminus of cAspAT (pcAspAT), and a pmAspAT variant in which the presequence and the amino-terminal domain of the mature enzyme are deleted (delta 2-57 pmAspAT). In RRL, delta 2-28 pmAspAT folds somewhat faster than intact pmAspAT, whereas the truncated delta 2-57 pmAspAT is unable to fold. In contrast, cAspAT and pcAspAT both fold with extreme rapidity. After synthesis in WGE, pmAspAT and delta 2-28 pmAspAT never acquire a protease-resistant conformation, whereas the folding of cAspAT and pcAspAT still occurs rapidly. We conclude that the presequence has only a minor role in determining the folding rate of the pmAspAT mitochondrial precursor protein in RRL or WGE and has no influence on the folding of the homologous cAspAT. Rather, the primary sequence of the mature part of the protein seems to dictate whether or how molecular chaperones regulate folding events.

Enzyme immunoassay in which a myeloma protein is used for detection of salmonellae.

An enzyme immunoassay (EIA) in which an immunoglobulin A monoclonal antibody from a myeloma (MOPC 467) is used was developed to detect the presence of Salmonella organisms. This myeloma protein binds to a flagellar determinant of the organisms but is not directed toward the H antigens. Of 100 strains tested, 94% were detectable with this antibody. The EIA, used with MOPC 467, is quick, sensitive, and specific, showing virtually no cross-reactivity to other enteric organisms. Initial screening of antibody reactivity was performed by Ouchterlony gel diffusion with the supernatants of heat-treated Salmonella cultures. After this, an EIA was performed on the heat extracts with the myeloma protein, which had been directly coupled to alkaline phosphatase. A positive reaction was indicated by the production of a yellow color after the addition of a substrate (p-nitrophenylphosphate), and this was quantitated by determining the absorbance at 405 nm. The EIA proved to be slightly more sensitive than the Ouchterlony analysis. The sensitivity of the EIA is such that as few as 10(6) Salmonella organisms per ml were detected. This concentration was easily obtained after a 24-h preenrichment incubation of the sample. Mixtures of Salmonella strains with a 10 x concentration of Escherichia coli did not prevent detection of the Salmonella strains. This EIA can be successfully used to detect contamination of foods, as it was used to detect the intentional contamination of infant formula in these studies. Indications are that the EIA is sensitive enough to detect Salmonella strains in M broth subcultures taken directly from a preenrichment culture. Testing of samples could thus be completed 36 h after culture initiation, rather than after 96 h, the time currently needed.

Effects of heating on the ion-gating function and structural domains of the acetylcholine receptor.

The ion-gating ability and the protein electrophoretic band patterns of the acetylcholine receptor from Torpedo californica electroplax were examined after receptor-enriched membrane vesicles were progressively heated. The ion translocation function was lost over a temperature range of 40-55 degrees C. Previous results have shown that the stoichiometry of alpha-bungarotoxin binding is not affected by these temperatures, although bound toxin reversibly dissociates within this temperature range, and that toxin binding is irreversibly lost at somewhat higher temperatures [Soler, G., Farach, M.C., Farach, H. A., Jr., Mattingly, J.R., Jr., & Martinez-Carrion, M. (1983) Arch. Biochem. Biophys. 225, 872]. Thermal gel analysis [Lysko, K. A., Carlson, R., Taverna, R., Snow, J., & Brandts, J.F. (1981) Biochemistry 20, 5570], a sodium dodecyl sulfate-polyacrylamide gel electrophoretic procedure which detects thermally induced aggregation of the components of multimeric systems, was applied to heated acetylcholine receptor enriched membranes. This technique suggests two structural domains susceptible to thermal perturbation within the receptor molecule, one consisting of the Mr 50 000 and the two Mr 40 000 subunits and the other consisting of the Mr 60 000 and 65 000 subunits. Heat disrupts molecular events linking agonist binding with ion-channel opening in the acetylcholine receptor molecule.

Properties of the active site lysyl residue of mitochondrial aspartate aminotransferase in solution.

Two vitamin B6 derivatives, N-bromoacetylpyridoxamine (BAPM) and its phosphate ester have been found to be affinity-labeling reagents for mitochondrial aspartate aminotransferase (EC 2.6.1.1). These derivatives were first shown to react with a critical sulfhydryl group in tryptophan synthase (Higgins, W., and Miles, E. W. (1978) J. Biol. Chem. 253, 4648-4652). In the apoaminotransferase, BAPM has now been found to inactivate by covalently modifying a critical lysyl residue, preventing reconstitution of the apoenzyme by pyridoxal 5'-phosphate. The dependence of the rate of inactivation upon the concentration of the reagent is consistent with a rapid equilibrium binary complex formation prior to the inactivation reaction. Both the dissociation constant for this complex and the rate of the reaction leading to inactivation are dependent on pH. BAPM binds best from pH 7.5 to 8.5. The rate of inactivation increases from pH 6 to 9. Succinate and phosphate competitively bind to the apoenzyme, protecting against BAPM inactivation. The C-5'-phosphorylated derivative is rapidly and tightly bound by the apotransaminase to form an inactive, noncovalent adduct. This bound reagent subsequently alkylates Lys-258. The rate of this covalent incorporation increases from pH 6 to 9 and is greater than the rate of BAPM modification at all pH values. The effect of pH on the reaction rates of both pyridoxal derivatives is interpreted to indicate protonation of Lys-258 at neutral pH values. These derivatives may also be analogs to a reaction intermediate different from those observed in other affinity-labeling studies. The ionization states of the Lys-258 epsilon-amino group apparently vary with the nature of the affinity label. These variations can be explained in terms of changing ionization states of Lys-258 in the steps of catalysis as well as in terms of the occupancy of charged sites on the protein by active site-directed substrates or inhibitory compounds.