Effect of the rehydration medium on the recovery of freeze-dried lactic acid bacteria.

Sixteen cultures of lactic acid bacteria were freeze-dried in 10% nonfat skim milk plus 0.75 M adonitol and rehydrated by using different rehydration media. Marked variations in their capacity to repair cellular damage after freeze-drying were observed among the species and strains under consideration.     

Membrane and cytoskeletal changes associated with IgE-mediated serotonin release from rat basophilic leukemia cells

Binding of antigen to IgE-receptor complexes on the surface of RBL-2H3 rat basophilic leukemia cells is the first event leading to the release of cellular serotonin, histamine, and other mediators of allergic, asthmatic, and inflammatory responses. We have used dinitrophenol-conjugated bovine serum albumin (DNP-BSA) as well as the fluorescent antigen, DNP-B-phycoerythrin, and the electron-dense antigen, DNP-BSA-gold, to investigate dynamic membrane and cytoskeletal events associated with the release of [3H]serotonin from anti-DNP-IgE-primed RBL-2H3 cells. These multivalent antigens bind rapidly to cell surface IgE-receptor complexes. Their distribution is initially uniform, but within 2 min DNP-BSA-gold is found in coated pits and is subsequently internalized. Antigen internalization occurs in the presence and absence of extracellular Ca2+. The F-actin content of the detergent-extracted cell matrices analyzed by SDS PAGE decreases during the first 10-30 s of antigen binding and then increases by 1 min to almost double the control levels. A rapid and sustained increase is also observed when total F-actin is quantified by flow cytometry after binding of rhodamine-phalloidin. The antigen-stimulated increase in F-actin coincides with (and may cause) the transformation of the cell surface from a finely microvillous to a highly folded or plicated topography. Other early membrane responses include increased cell spreading and a 2-3-fold increase in the uptake of fluorescein-dextran by fluid pinocytosis. The surface and F-actin changes show the same dependence on DNP-protein concentration as stimulated [3H]serotonin release; and both the membrane responses and the release of mediators are terminated by the addition of the non-cross-linking monovalent ligand, DNP-lysine. These data indicate that the same antigen-stimulated transduction pathway controls both the membrane/cytoskeletal and secretory events. However, the membrane and actin responses to IgE-receptor cross-linking are independent of extracellular Ca2+ and are mimicked by phorbol myristate acetate, whereas ligand-dependent mediator release depends on extracellular Ca2+ and is mimicked by the Ca2+ ionophore A23187.     

TRH and TRH-OH in the pancreas of adult and newborn rats

TRH and its metabolite TRH-OH have been measured by specific radioimmunoassays in acid extracts of pancreas in adults and developing rats. TRH and TRH-OH immunoreactivity had the same ontogenic pattern with a maximal concentration on day 4 followed by a progressive return towards adult levels on day 20. A significant linear correlation was found between TRH levels and the TRH/TRH-OH ratio. The range of TRH/TRH-OH ratio varied from 136 +/- 1.6, at the peak of concentrations of both peptides, to 18 +/- 3.9 on day 20. Pancreatic TRH and TRH-OH had the same elution pattern as corresponding synthetic peptides both on Biogel P2 and high-pressure liquid chromatography. The origin of TRH-OH as well as its potential function need further investigations.     

Kinetic studies on formation of cytochrome oxidase of Rhodopseudomonas capsulata after a shift from phototrophic to chemotrophic growth.

Rhodopseudomonas capsulata cells were shifted from phototrophic (anaerobic, light) to chemotrophic (semiaerobic, dark, 10% air saturation) growth conditions. During the adaptation period of 4 h, the bacteriochlorophyll content of cells and membranes decreased, and a newly synthesized 65-kilodalton polypeptide of the cytochrome oxidase was incorporated into the membrane fraction. The enzymatic activity of the cytochrome oxidase increased strongly after a lag time of 2 h. The amount of cytochrome oxidase protein does not follow the same kinetics. The relative amount of a membrane-bound cytochrome c of low molecular weight, which has been proposed to be a donor for the cytochrome oxidase, increased during adaptation.     

Fusion of liposomes and chromatophores of Rhodopseudomonas capsulata: effect on photosynthetic energy transfer between B875 and reaction center complexes.

The photosynthetic chromatophore membranes of Rhodopseudomonas capsulata were fused with liposomes to investigate the effects of lipid dilution on energy transfer between the bacteriochlorophyll-protein complexes of this membrane. Phosphatidylcholine-containing liposomes were mixed with chromatophores at pH 6.0 to 6.2, and the mixture was fractionated on discontinuous sucrose gradients into four membrane fractions with lipid-to-protein ratios that varied 11-fold. Freeze-fracture electron microscopy revealed that the fractions contained closed vesicles formed by the fusion of liposomes to chromatophores. Particles with 9-nm diameters on the P fracture faces did not appear to change in size with increasing lipid content, but the number of particles per membrane area decreased proportionally with increases in the lipid-to-protein ratio. The bacteriochlorophyll-to-protein ratios, electrophoretic polypeptide profiles on sodium dodecyl sulfate-polyacrylamide gels, and light-induced absorbance changes at 595 nm caused by photosynthetic reaction centers were not altered by fusion. The relative fluorescence emission intensities due to the B875 light-harvesting complex increased significantly with increasing lipid content, but no increases in fluorescence due to the B800-B850 light-harvesting complex were observed. Electron transport rates, measured as succinate-cytochrome c reductase activities, decreased with increased lipid content. The results indicate an uncoupling of energy transfer between the B875 light-harvesting and reaction center complexes with lipid dilution of the chromatophore membrane.     

Interaction of Opiates with Opioid Binding Sites in the Bovine Adrenal Medulla: I. Interaction with δ and μ Sites

In the present study we examined the interaction of opiates with the delta and mu opioid binding sites in the bovine adrenal medulla. [3H][D-Ala2, D-Leu5]-enkephalin ( [3H]DADLE) in the presence of saturating concentrations of morphiceptin was used to analyze delta site interactions, whereas either [3H]DADLE in the presence of saturation concentrations of [D-Ser2, Leu5]-enkephalin-Thr6 (DSLET) or [3H][D-Ala2, Me-Phe4, Gly5-ol]-enkephalin ( [3H]DAGO) was used for the determination of mu sites. Both binding sites were found to interact stereoselectively with opiates. The binding was affected differentially by proteolytic enzymes (trypsin, alpha-chymotrypsin, pepsin), N-ethylmaleimide, and A2-phospholipase. Kinetic and equilibrium binding studies revealed that in each case radiolabeled opiates interact with one class of binding sites, following simple second-order bimolecular kinetics. Competition for binding by opiates and opioid peptides confirmed the delta and mu selectivity of these sites. Monovalent (Na+, Li+, K+) and divalent (Mg2+, Mn2+, Ca2+) ions interacted differentially with these two binding sites: In general, monovalent cations affected preferentially the apparent number of binding sites, whereas divalent ions modified the equilibrium dissociation constant. Furthermore, positive or negative cooperativity and an apparent heterogeneity of binding sites were detected under some ionic conditions.     

The presence and photoregulation of protochlorophyllide reductase in green tissues

Summary The enzyme protochlorophyllide (pchlide) reductase has been identified amongst the peptides, resolved by sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE), of chloroplast membranes from oat and barley plants. In support of this identification the enzymic activity associated with the enzyme has also been measured in the same preparations. A higher level of enzyme was found in plants which had been darkened prior to extraction. Based on this data, mechanisms for the light regulated diurnal variation of the reductase are discussed.     

Isolation and characterization of Pediococcus halophilus from salted anchovies (Engraulis anchoita)

The presence of bacteria in salted anchovies during and at the end of the curing process was investigated. Attempts to isolate bacteria under aerobic or anaerobic conditions led to the isolation of only bacteria of the genus Pediococcus which were identified as Pediococcus halophilus. The isolates correspond to a rather heterogeneous group in which some of the members differ in some biochemical tests from the types described in the literature.     

Differential distribution of3H dihydrotestosterone and3H estradiol nuclear binding sites in mouse male accessory sex organs

Summary The distribution of specific nuclear binding sites for androgens and estrogens in the male accessory sex organs of the mouse was assessed by autoradiography with³H dihydrotestosterone (³H DHT) and³H estradiol (³H E₂). With³H DHT nuclear labeling differed among the epithelia of the organs. It was high in seminal vesicle and ampullary gland, moderate in ventral prostate, urethral gland, prostatic excretory ducts and the ampulla ductus deferentis, low in dorsal prostate and low or absent in coagulation gland. With³H E₂, in contrast, epithelial nuclear labeling was high only in coagulation gland, moderate or low in seminal vesicle, low or absent in ventral and dorsal prostate and absent in ampullary gland and ampulla ductus deferentis. In the lamina propria of all organs nuclear labeling with³H DHT was generally moderate and existed only in some cells, with the highest number in the ampulla ductus deferentis. With³H E₂, nuclear labeling in the lamina propria showed a high intensity in all organs, except in ventral and dorsal prostate which remained unlabeled. Many labeled cells were found in the deferent duct and its ampulla, while in the other organs only a few cells showed nuclear labeling with³H E₂. In the smooth muscle sheath of all organs, some muscle cells were moderately labeled with³H DHT, but not with³H E₂. The results indicate the presence of nuclear receptors in male accessory sex organs for both dihydrotestosterone and estradiol. The differential patterns of³H DHT and³H E₂ nuclear uptake suggest differential sensitivities of the individual organs and their tissue compartments for androgens and estrogens. Supported by PHS grant NSO9914 to W.E.S. and Deutsche Forschungsgemeinschaft Dr94/4 to U.D. The work of Dr. Schleicher and his stay in Chapel Hill were additionally sponsored by Studienstiftung des Deutschen Volkes and Boehringer-Ingelheim Fonds