Immunocytochemical detection of the growth-associated protein B-50 by newly characterized monoclonal antibodies in human brain and muscle

The growth-associated protein B-50 also termed GAP-43, F1, pp46, P-57 and neuromodulin is a nervous tissuespecific protein kinase C (PKC) substrate that is considered to play a major role in neurite formation, regeneration, and neuroplasticity. We describe the isolation of seven mouse monoclonal antibodies (Mabs) directed against B-50. The Mabs are produced against the bovine B-50, selected by ELISA for cross-reactivity with its human counterpart, and evaluated on Western blots in comparison with the well-characterized affinity-purified rabbit polyclonal antibodies to rat-B-50. The Western blots show that the Mabs NM1, NM4, and NM6 recognize specifically the B-50 of bovine, human, and rat brain extract and the purified PKC phosphorylated and unphosphorylated rat B-50 isoforms. The Mabs NM2 and NM3 cross-react with bovine B-50 immunoreactive c-kinase substrate (BICKS), a protein sharing a 17 amino acid sequence homology with B-50. Two Mabs are useful for the detection of B-50 immunoreactivity in formalin-fixed human and rat brain tissues. In human specimen of the hippocampus, a characteristic neuropil distribution of B-50 is detected by the Mabs. In human muscle, Mabs reveal B-50 in nerve bundles and in axons at motor end plates. Thus, these Mabs are useful in investigating the function and localization of the B-50 protein.     

Heat treatment results in a loss of transgene-encoded activities in several tobacco lines.

Heat treatment (37 degrees C) of transgenic tobacco (Nicotiana tabacum) plants led to a reversible reduction or complete loss of transgene-encoded activities in about 40% of 10 independent transformants carrying the luciferase-coding region fused to the 355 cauliflower mosaic virus or the soybean small subunit promoter and the nopaline synthase promoter driving the neomycin phosphotransferase gene, whereas the other lines had temperature-tolerant activities. Temperature sensitivity or tolerance of transgene-encoded activities was heritable. In some of the lines, temperature sensitivity of the transgene-encoded activities depended on the stage of development, occurring in either seedlings (40% luciferase and 50% neomycin phosphotransferase) or adult plants (both 40%). The phenomenon did not correlate with copy numbers or the homo- or hemizygous state of the transgenes. In lines harboring a temperature-sensitive luciferase activity, reduction of bioluminescence was observed after 2 to 3 h at 37 degrees C. Activity was regained after 2 h of subsequent cultivation at 25 degrees C. Irrespective of the reaction to the heat treatment, the level of luciferase RNA was slightly increased at 37 degrees C. Only in lines showing temperature sensitivity of transgene-encoded activities was the amount of luciferase and neomycin phosphotransferase strongly reduced. In sterile culture, heat treatment for 15 d did not cause visible damage or changes in plant morphology. In all plants tested a slight induction of the heat-shock response was observed at 37 degrees C.     

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.     

On feeding and nutrition in Fungiacava eilatensis (Bivalvia, Mytilidae), a commensal living in fungiid corals

The enlarged inhalant siphon of Fungiacava eilatensis opens into the coelenteron of species of fungiid corals with which it lives in commensal association. Material consisting of mucus, zooxanthellae, nematocysts, plankton and inorganic matter, is taken exclusively from the coelenteron. The very mobile foot possibly assists in food collection and in the removal of pseudofaeces; but, with large ctenidia, the bivalve is a typical ciliary feeder. Experiments with labelled zooxanthellae reveal that these are taken into the gut of Fungiacava with subsequent metabolic incorporation of products derived from them. The other prime source of food must be phytoplankton carried in with the feeding currents of the coral, itself carnivorous so that there is no competition for food between commensal and host. The Fungia zooxanthella– Fungiacava association operates as a "Troika" the productivity of which is autoregulated in proportion to the number of bivalves present. The inorganic wastes of the bivalve (as well as those of the coral) are utilized by the zooxanthellae, resultant increase in the algal component becoming available as food to the bivalve. Losses in the cycle are balanced by intake of exogenous food.     

SPATIAL POPULATION STRUCTURE IN THE WHIRLIGIG BEETLE DINEUTUS ASSIMILIS: EVOLUTIONARY INFERENCES BASED ON MITOCHONDRIAL DNA AND FIELD DATA

The spatial population structure of the pond-living water beetle Dineutus assimilis (Coleoptera: Gyrinidae) was investigated through a field study of population dynamics and dispersal, with a concurrent assessment of the spatial distribution of mitochondrial DNA (mtDNA) restriction-fragment-length polymorphism (RFLP). A comprehensive 2-yr survey within a 60-km² study area revealed pronounced fluctuations in local abundances, including extinctions and colonizations. The recapture of marked individuals showed that dispersal among ponds is frequent in both males and females and connects populations on a large geographic scale (maximum observed flight distance: 20 km). The population structure of D. assimilis is thus characterized by both pronounced genetic drift and frequent gene flow. Together, these two forces generate a pattern of very local and transient genetic differentiation. Mitochondrial DNA samples collected within a few kilometers indicate highly significant spatial structure, if newly founded demes or those that experienced recent bottlenecks are included. These results based on four demes within the study area were placed into a regional context by further samples collected at distances of 100 km and 200 km. F_st estimates computed on increasing spatial scales were variable but showed no increasing trend. Thus, gene flow exerts a strong homogenizing force over a wide geographic range but is counteracted locally by genetic drift. These findings highlight the need to supplement estimates of F_st with additional data to arrive at valid interpretations of the genetic information. More generally, this study raises questions about how to capture the relevant features of dynamic, subdivided populations to understand their evolutionary dynamics.     

Phosphorylation of the acetylcholine receptor by protein kinase C and identification of the phosphorylation site within the receptor delta subunit

Purified acetylcholine receptor is rapidly and specifically phosphorylated by partially purified protein kinase C, the Ca2+/phospholipid-dependent enzyme. The receptor delta subunit is the major target for phosphorylation and is phosphorylated on serine residues to a final stoichiometry of 0.4 mol of phosphate/mol of subunit. Phosphorylation is dose-dependent with a Km value of 0.2 microM. Proteolytic digestion of the delta subunit phosphorylated by either protein kinase C or the cAMP-dependent protein kinase yielded a similar pattern of phosphorylated fragments. The amino acids phosphorylated by either kinase co-localized within a 15-kDa proteolytic fragment of the delta subunit. This fragment was visualized by immunoblotting with antibodies against a synthetic peptide corresponding to residues 354-367 of the receptor delta subunit. This sequence, which contains 3 consecutive serine residues, was recently shown to include the cAMP-dependent protein kinase phosphorylation site (Souroujon, M. C., Neumann, D., Pizzighella, S., Fridkin, M., and Fuchs, S. (1986) EMBO J. 5, 543-546). Concomitantly, the synthetic peptide 354-367 was specifically phosphorylated in a Ca2+- and phospholipid-dependent manner by protein kinase C. Furthermore, antibodies directed against this peptide inhibited phosphorylation of the intact receptor by protein kinase C. We thus conclude that both the cAMP-dependent protein kinase and protein kinase C phosphorylation sites reside in very close proximity within the 3 adjacent serine residues at positions 360, 361, and 362 of the delta subunit of the acetylcholine receptor.