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.     

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.     

Solid-Supported Oligonucleotide Systems for Special Biomedical Applications

Abstract

The use of composite beads consisting of a 6 μm polystyrene core with 30 nm surface-bound silica particles to routine automatic oligodeoxynucleotide (ODN) synthesis is described.     

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.     

Murine gamma interferon inhibits v-mos-induced fibroblast transformation via down regulation of retroviral gene expression.

Expression of the retroviral vector Neor myeloproliferative sarcoma virus (MPSV), which contains the v-mos oncogene and the neomycin resistance gene, leads to neoplastic transformation of mouse fibroblasts. Murine recombinant gamma interferon (IFN-gamma) could revert the neoplastic properties of established Neor MPSV-transformed cell lines to an apparently untransformed phenotype. In the presence of IFN-gamma, the Neor MPSV transformants showed a greater than 97% reduction of cloning efficiency in soft agar, strongly reduced proliferative capacity, and morphological changes. The IFN-gamma-induced phenotypic reversion was preceded by a rapid and selective reduction of all retroviral RNA species, apparently due to IFN-gamma action on the long terminal repeat of Neor MPSV. The mRNA levels of cellular genes either remained unaffected (beta-actin) or were even enhanced (H-2) in IFN-gamma-treated Neor MPSV-transformed cells. Upon removal of IFN-gamma, retroviral gene expression was fully recovered and a gradual reappearance of the transformed phenotype of these cells within 3 weeks was noted. These data show that IFN-gamma can cause a virtually complete, but reversible, inhibition of v-mos-induced neoplastic properties in transformed fibroblasts by selective down regulation of retroviral RNA levels.     

Methyljasmonate and α-linolenic acid are potent inducers of tendril coiling

A coiling-inducing factor was isolated from tendrils of Bryonia dioica Jacq. and identified by infrared, ¹H-, ¹³C-nuclear magnetic resonance and mass spectrometry as -linolenic acid. When applied to detached tendrils, exogenous -linolenic acid, but not linoleic acid or oleic acid, induced tendril coiling. Further investigations showed that metabolites of -linolenic acid, jasmonic acid and, even more so, methyljasmonate, are highly effective inducers of tendril coiling in B. dioica. Methyljasmonate was most active when administered by air and, in atmospheric concentrations as low as 40–80 nM, induced a full free-coiling response with kinetics similar to mechanical stimulation. Even atmospheric levels as low as 4–5 nM methyljasmonate were still found to be significantly active. Methyljasmonate could be one of the endogenous chemical signals produced in mechanically stimulated parts of a tendril and, being highly volatile, act as a diffusible gaseous mediator spreading through the intracellular spaces to trigger free coiling of tendrils.Abbreviations EI-MS electron impact-mass spectrometry - HPLC high-performance liquid chromatography - IAA indole-3-acetic acid - NMR nuclear magnetic resonance - TFA trifluoroacetic acid We are indebted to the Deutsche Forschungsgemeinschaft, Bonn and the Fonds der Chemischen Industrie, Frankfurt (literature provision) for their support and to Dr. C. Brückner, Halle, for jasmonic-acid determinations.     

Transfer and Expression of Lithoautotrophy and Denitrification in a Host Lacking These Metabolic Activities

The conjugative 450-kilobase-pair megaplasmid pHG1 from Alcaligenes eutrophus H16 was transferred to the herbicide-degrading soil bacterium A. eutrophus JMP134. This transfer was achieved by means of RP4 mobilization and a Tn5-Mob insertion provided in trans on the megaplasmid replicon. Although kanamycin-resistant transconjugants also occurred with other gram-negative species such as Rhizobium, Agrobacterium, and thiobacteria, A. eutrophus JMP134 was the only recipient which stably maintained the megaplasmid. pHG1-containing transconjugants derived from JMP134 expressed all metabolic functions associated with the plasmid: the ability to oxidize hydrogen through catalysis of two hydrogenases, to assimilate carbon dioxide via the Calvin cycle pathway, and to grow with nitrate anaerobically. All of these metabolic activities were absent in the original strain JMP134.     

Bioluminescence of Marine Dinoflagellates : I. An underwater photometer for day and night measurements

Portable light-baffled underwater photometers have been designed for the measurement of dinoflagellate bioluminescence by day and night. Maximal light emission is obtained by mechanical stimulation in a defined volume. The pump which stimulates the dinoflagellates also constantly replenishes the sample volume so that continuous measurements are possible. Evidence for both diurnal variation and vertical migration is presented. Using luminous bacteria for calibration a single dinoflagellate has been found to emit of the order of 10¹⁰ light quanta per flash. The technique suggests that large scale mapping of bioluminescence is feasible.     

Isolation of the vitellogenin-binding protein from locust ovaries

A rapid, efficient procedure for the isolation and purification of the vitellogenin binding protein from locust ovarian membranes is described. After solubilization with the nonionic detergent octyl-β-D-glucoside and removal of the detergent, the binding protein is subjected to affinity chromatography on vitellogenin coupled covalently to Affi-Gel 15. The binding protein is eluted with suramin and EDTA at low pH value. Sodium dodecyl sulfate (SDS) polyacrylamide gel electrophoresis reveals a polypeptide with a molecular weight of 156,000 in the eluted fraction. By ligand blotting this polypeptide could be identified as the vitellogenin binding protein. It retains its high-affinity binding properties. The specific binding of vitellogenin increases from 4.8 μg (intact ovarian membranes) to 170.9 μg (affinity purified binding protein) per mg membrane protein, which corresponds to a purification factor of 35.     

Immunocytochemistry of 5-bromo-2′-deoxyuridine labelled oligonucleotide probes

Summary A synthetic oligonucleotide probe, complementary to oxytocin m-RNA was labelled enzymatically with 5-bromo-2-deoxyuridine (5-BrdU) and, with [-³²P]-ATP. The labelled probes were used for in situ, hybridization of histological sections of the mouse hypothalamus. A monoclonal antibody to 5-BrdU and the streptavidine-peroxidase technique were used in order to visualize hybridization with the 5-BrdU labelled probe. In situ hybridization with [³²P] labelling was detected autoradiographically. With both methods hybridized neurons were visible in the magnocellular hypothalamic nuclei. While immunostaining and radio-labelling provided similar localization of oxytocin m-RNA, only the immunocytochemical technique showed clear cellular resolution of the reaction product. In situ hybridization with 5-BrdU labelled probes followed by 5-BrdU immunocytochemistry seems to be a powerful alternative to common autoradiographic techniques.