Startling Sweet Temptations: Hedonic Chocolate Deprivation Modulates Experience,Eating Behavior,and Eyeblink Startle

Many individuals restrict their food intake to prevent weight gain. This restriction has both homeostatic and hedonic effects but their relative contribution is currently unclear. To isolate hedonic effects of food restriction, we exposed regular chocolate eaters to one week of chocolate deprivation but otherwise regular eating. Before and after this hedonic deprivation, participants viewed images of chocolate and images of high-calorie but non-chocolate containing foods, while experiential, behavioral and eyeblink startle responses were measured. Compared to satiety, hedonic deprivation triggered increased chocolate wanting, liking, and chocolate consumption but also feelings of frustration and startle potentiation during the intertrial intervals. Deprivation was further characterized by startle inhibition during both chocolate and food images relative to the intertrial intervals. Individuals who responded with frustration to the manipulation and those who scored high on a questionnaire of impulsivity showed more relative startle inhibition. The results reveal the profound effects of hedonic deprivation on experiential, behavioral and attentional/appetitive response systems and underscore the role of individual differences and state variables for startle modulation. Implications for dieting research and practice as well as for eating and weight disorders are discussed.     

Cell surface distribution and intracellular fate of human beta-very low density lipoprotein in cultured peritoneal mouse macrophages: a cytochemical and immunocytochemical study

The receptor-mediated endocytosis pathway of colloidal gold labeled beta-very low density lipoprotein (beta-VLDL-Au) derived from patients with familial dysbetalipoproteinemia was analyzed at the ultrastructural level in macrophages. The results showed that beta-VLDL-Au complexes were specifically recognized by a cell surface receptor of the macrophages. beta-VLDL-Au particles once bound to the randomly distributed cell surface receptors clustered in coated pits and were taken up by coated vesicles. Subsequently, the beta-VLDL-Au particles passed through tubular structures and small endosomes before deposited into large electron lucent smooth surfaced endosomes. As revealed by ruthenium red and enzyme cytochemistry the endosomes appeared to be separated from the extracellular space and did not contain acid phosphatase. There were no clear signs of passage of beta-VLDL through the Golgi complex. The accumulation of many flocculated gold particles within Ac-Pase positive vesicles suggests that beta-VLDL once internalized by the macrophages is diverted into a degradative pathway. Incubation of beta-VLDL-loaded macrophages with the hydrophobic fluorescent dye nile red revealed numerous large fluorescent bodies within the cells indicating that the macrophages accumulate large amounts of lipid droplets with time. Additional studies large amounts of lipid droplets with time. Additional studies with native beta-VLDL in conjunction with postembedding immunocytochemical techniques were used to delineate further the intracellular pathway. Immunolabeling was carried out on thin sections of LR White embedded cells using affinity-purified polyclonal rabbit antibodies against apolipoprotein B with the protein A-gold or goat anti-rabbit IgG-gold technique. Indirect visualization of beta-VLDL by these immunocytochemical studies yielded results comparable to those with gold-labeled beta-VLDL. On the basis of both indirect immunocytochemical and direct cytochemical localization of beta-VLDL it is concluded that although colloidal gold labeling of beta-VLDL molecules unquestionably modifies their morphology, their function appears to be unaltered, at least with respect to the process of receptor-mediated endocytosis.     

Synchrony of bone marrow proliferation and maturation as the origin of cyclic haemopoiesis

Abstract. Cyclic haemopoiesis in Grey Collie dogs is characterized by stable oscillations in all haemopoietic lineages. It is proposed that in these animals, in contrast to normal animals, the maturation process of haemopoietic (in particular granuloid) cells from the primitive progenitors to the functional cells is characterized by an abnormally strong synchrony. It is conjectured that the marrow maturation time has a very small variance compared with non-cyclic normal dogs. With a mathematical model of haemopoiesis it is shown that small fluctuations are amplified via regular feedback processes such that stable granuloid oscillations are established. Erythroid oscillations are induced indirectly by granuloid feedback to the stem cell pool. The model calculations further show that the synchrony hypothesis of bone marrow maturation can quantitatively explain the following experimental results: (1) the maintenance of stable cycles of granuloid and erythroid bone marrow and blood cells with a period of approximately 14 d; (2) the disappearance of granuloid and erythroid cycles during the administration of the colony stimulating factor rhG-CSF; (3) the reappearance of oscillations when the administration of CSF is discontinued; (4) the cessation of cycles during endotoxin application; and (5) the persistence of cycles during erythroid manipulations (bleeding anaemia, hypoxia, hypertransfusion). We therefore conclude that cyclic haemopoiesis is not caused by a defect in the regulatory control system but by an unusual maturation process.     

Limited Tryptic Proteolysis of the Benzodiazepine Binding Proteins in Different Species Reveals Structural Homologies

Peptide mapping can be used to elucidate further the structural similarities of the benzodiazepine binding proteins in different vertebrate species. Crude synaptic membrane preparations were photoaffinity-labeled with [3H]flunitrazepam and subsequently degraded with various concentrations of trypsin. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis followed by fluorography allowed a comparison of the molecular weights of photolabeled peptides in different species. Tryptic degradation led to a common peptide of 40K in all species investigated, a finding indicating that the benzodiazepine binding proteins are structurally homologous in higher bony fishes and tetrapods.     

Ribosomal frameshifting in plants: a novel signal directs the -1 frameshift in the synthesis of the putative viral replicase of potato leafroll luteovirus.

The 5.8 kb RNA genome of potato leafroll luteovirus (PLRV) contains two overlapping open reading frames, ORF2a and ORF2b, which are characterized by helicase and RNA polymerase motifs, respectively, and possibly represent the viral replicase. Within the overlap, ORF2b lacks an AUG translational start codon and is therefore presumably translated by -1 ribosomal frameshifting as a transframe protein with ORF2a. This hypothesis was studied by introducing the putative frameshift region into an internal position of the beta-glucuronidase (GUS) gene and testing for the occurrence of frameshifting in vivo by transient expression of GUS activity in potato protoplasts as well as in vitro by translation in the reticulocyte system. Both experimental approaches demonstrate that a -1 frameshift occurs at a frequency of approximately 1%. Site-directed mutagenesis identified the frameshift region and the involvement of the novel heptanucleotide motif UUUAAAU in conjunction with an adjacent stem-loop structure. Part of this stem-loop encodes a basic region in the ORF2b moiety of the transframe protein which was shown by binding experiments with PLRV RNA to represent a nucleic acid-binding domain. These data support a possible biological significance of the frameshift to occur at this position of the large overlap by including the putative RNA template-binding site of the PLRV replicase in the ORF2a/ORF2b transframe protein.     

Sensitive chemiluminescent detection of digoxigenin-labeled nucleic acids: a fast and simple protocol and its applications.

A fast and simple protocol for the chemiluminescent detection of digoxigenin-labeled nucleic acids with anti-digoxigenin antibody Fab fragments coupled to alkaline phosphatase and 3-(4-methoxyspiro[1,2-dioxetane-3,2'-tricyclo-[3.3.1.1 (3,7)]decan]-4- yl)phenyl phosphate as substrate is described. The washing and blocking procedure was optimized to yield low background even on positively charged nylon membranes. The sensitivity of the system is equal or better than radioactive methods. Exposure to x-ray or Polaroid film for up to 30 minutes is sufficient for the detection of 70 femtograms of homologous DNA. Human single-copy genes are detected in Southern blots of as low as 0.3 microgram total placental DNA. Blots can be reprobed multiple times very easily. The advantages of the digoxigenin system are high sensitivity, absence of background and ease of reprobing and are illustrated by applications for single-copy gene detection in genomic blots of human DNA, Northern hybridizations to rare mRNA, detection of E. coli genes on blots of genomic digests after pulse field gel electrophoresis, as well as for nonradioactive DNA sequencing blots with digoxigenin-labeled primers.     

Effects of spines and thorns on Australian arid zone herbivores of different body masses

Summary We investigated the effects of thorns and spines on the feeding of 5 herbivore species in arid Australia. The herbivores were the rabbit (Oryctolagus cuniculus), euro kangaroo (Macropus robustus), red kangaroo (Macropus rufus), sheep (Ovis aries), and cattle (Bos taurus). Five woody plants without spines or thorns and 6 woody plants with thorns were included in the study. The spines and thorns were not found to affect the herbivores' rates of feeding (items ingested/min), but they did reduce the herbivores' rates of biomass ingestion (g-dry/item). The reduction in biomass ingested occurred in two ways: at a given diameter, twigs with spines and thorns had less mass than undefended plants, and the herbivores consumed twigs with smaller diameters on plants with spines and thorns. The relative importance of the two ways that twigs with spines and thorns provided less biomass varied with herbivore body mass. Reduced twig mass was more important for small herbivores, while large herbivores selected smaller diameters. The effectiveness of spines and thorns as anti-herbivore defenses did not vary with the evolutionary history of the herbivores (i.e. native vs. introduced). Spines and thorns mainly affected the herbivores' selection of maximum twig sizes (reducing diameter and mass), but the minimum twig sizes selected were also reduced.     

Distribution and immunolocalization of stachyose synthase in Cucumis melo L.

Indirect evidence for the site of stachyose biosynthesis has been provided by determining the occurrence and distribution of stachyose, raffinose and galactinol, the donor of the galactosyl moiety for stachyose synthesis, in Cucumis melo L. cv. Ranjadew. Studies of enzyme activities for the synthesis of these sugars and their distribution in different plant organs and isolates has led to the conclusion that stachyose is synthesized mainly in mature leaves and seeds. Nevertheless, stachyose-synthase activity varied with leaf age, the developmental stage of a plant, the growing season and the plant cultivar used. No stachyose or stachyose-synthase activity could be detected in isolated mesophyll protoplasts and chloroplasts, whereas both were found in a minor-vein-enriched fraction isolated from mature leaves. The conclusion that stachyose biosynthesis is associated with minor veins was confirmed by immunolocalization of the enzyme. Positive specific immunoreactivity of stachyose synthase with polyclonal anti-stachyose-synthase antibodies, labeled with protein A-gold, was detected in intermediary cells of leaf minor veins. The implication of this local synthesis of the main transport sugar for phloem loading in mature leaves of Cucumis melo is discussed.Abbreviation RUBPCase ribulose-1,5-bisphosphate carboxylase This work was supported by Deutsche Forschungsgemeinschaft. The excellent assistance of Ms. B. Müller in preparing the samples for electron microscopy is gratefully acknowledged. The authors thank Professor H.J. Schneider-Poetsch for anti-RuBPCase antibodies.     

Stachyose synthesis in mature leaves of Cucumis melo. Purification and characterization of stachyose synthase (EC 2.4.1.67)

Galactinol: raffinose-6-galactosyltransferase (EC 2.4.1.67), a stachyose synthase, was extracted from mature leaves of Cucumis melo cv. Ranjadew and was purified to homogeneity by (NH₄)₂SO₄ precipitation, ion-exchange chromatography, gel-filtration and non-denaturing polyacrylamide gel electrophoresis. A specific activity of 516 kat · mg^-1 and a 160-fold purification was achieved. The pH optimum of the enzyme reaction was found to be 6.8 in sodium-phosphate buffer, and the temperature optimum 32° C. The purified enzyme was very sensitive towards SH-poisons but its reaction was hardly affected by changes in the ion composition of the assay medium. The two-substrate enzyme was specific for galactinol and raffmose; uridine-diphosphate galactose and p-nitrophenyl--d-galactoside as well as melibiose were not accepted by the purified enzyme. Stachyose synthesis was competitively inhibited by concentrations >4 mM raffinose as well as 2.5 mM galactinol. The K _m values determined under non-saturating conditions were 3.3 mM for raffinose and 7.7 mM for galactinol. Myoinositol was a strong competitive inhibitor with a K _i of 1.8mM. Galactinol was hydrolyzed in the absence of raffinose with a K _m of 0.8 mM. The pure enzyme is a protein with a molecular weight of at least 95 kDa and an isoelectric point of 5.1. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis showed the presence of two subunits of 45 and 50 kDa. Polyclonal antibodies from rabbit were obtained which were specific for the native enzyme but cross-reacted with other proteins separated under denaturing conditions.Abbreviations DEAE diethylaminoethyl - DTT dithiothreitol - FPLC fast protein liquid chromatography - HPLC high-performance liquid chromatography - PAGE polyacrylamide gel electrophoresis - SDS sodium dodecyl sulfate This work was supported by Deutsche Forschungsgemeinschaft. The gift of galactinol by Dr. T. Schweizer (Nestlé, Switzerland) is gratefully acknowledged.