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.     

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.     

Nerve Growth Factor Effect on Adenosine Transport in Cultured Chromaffin Cells

Chromaffin cells both recently isolated or in culture present a high-affinity adenosine transporter with a Km value of 1 microM. When cells were exposed to nerve growth factor (NGF; 10 ng/ml), the adenosine transporter affinity decreased to 3 microM. This value was maintained from 3 days after plating to the end of the culture period. A change in the transport capacity was observed, with a significant increase (approximately 200-260%) in NGF-cultured cells throughout the period studied.     

Modelling mass transfer and agitator performance in multiturbine fermentors

A methodology for mathematically analyzing agitator performance and mass transfer in large multiturbine production fermentors is presented. The application of this approach provides a method for determining axial dissolved oxygen profiles under conditions of known mass transfer rates as a function of agitation-aeration characteristics. A stagewise approach is used which divides the fermentor into a series of mixing cells. This allows for each turbine and mixing cell to be individually optimized. The model also permits the determination of the mass transfer coefficient for each turbine based upon limited dissolved oxygen data. The primary limitation of this approach rests in the limited data and correlations available for multiturbine systems. The structure of the modelling approach can serve as a basis for testing single turbine correlations and adapting them to multiturbine systems. The step-by-step details of the mathematical analysis are presented and interpreted. A series of computer simulations demonstrate the effect of typical fermentor operating variables on the axial dissolved oxygen profile. Further simulations demonstrate the effect of modifying agitator blade numbers on the dissolved oxygen profile and agitator power requirement.     

Effect of secretagogues on chromogranin A synthesis in bovine cultured chromaffin cells. Possible regulation by protein kinase C.

Chromogranin A is a major component of storage granules in many different secretory cell types. After [35S]methionine labelling of proteins from cultured bovine chromaffin cells, chromogranin A was immunoprecipitated with specific antibodies, and the radioactivity incorporated into chromogranin A was determined and used as an index of its synthesis rate. Depolarization of cells with nicotine or high K+ evoked a Ca2+-dependent increase in chromogranin A synthesis, whereas muscarine, which does not evoke significant Ca2+ influx from bovine chromaffin cells, had no effect on chromogranin A synthesis. Forskolin, an activator of adenylate cyclase, affected neither the basal nor the nicotine-stimulated rate of chromogranin A synthesis. In contrast, 12-O-tetradecanoylphorbol 13-acetate (TPA), an activator of protein kinase C, significantly enhanced the incorporation of radioactivity into chromogranin A. Sphingosine, an inhibitor of protein kinase C, abolished both nicotine-stimulated and TPA-induced chromogranin A synthesis. In addition, long-term treatment of chromaffin cells with TPA decreased protein kinase C activity and inhibited the nicotine-stimulated chromogranin A synthesis. These results suggest that protein kinase C may play an important role in the control of chromogranin A synthesis.     

Transient expression of a Ca2+-activated Cl- current during development of quail sensory neurons

The expression of a calcium-activated chloride current (ICl(Ca)) was studied during the development of the sensory neurons of quail trigeminal ganglia. This current is expressed in 20% of the neurons by the 5th day of embryonic development; it can be found in nearly all neurons by the 7th day and subsequently disappears in half of them. Similar results were obtained with dorsal root ganglion neurons. The disappearance of ICl(Ca) in part of the sensory neurons during development is not due to a selective death of the neurons possessing this current and our results suggest that it is mediated by an interaction of the sensory neurons with their target tissue.     

Peripheral actin filaments control calcium-mediated catecholamine release from streptolysin-O-permeabilized chromaffin cells

Adrenal medullary chromaffin cells were permeabilized by treatment with a streptococcal cytotoxin streptolysin O (SLO) which generates pores of macromolecular dimensions in the plasma membrane. SLO did not provoke spontaneous release of catecholamines or chromogranin A, a protein marker of the secretory granule, showing the integrity of the secretory vesicle membrane. However, the addition of micromolar free calcium concentration induced the corelease of noradrenaline and chromogranin A, indicating that secretory products are liberated by exocytosis. Calcium-dependent exocytosis from SLO-permeabilized cells required Mg-ATP and could not occur in the presence of other nucleotides. The pores generated by the toxin were large enough to introduce proteins, e.g., immunoglobulins, but also caused efflux of the cytosolic marker lactate dehydrogenase. Despite this, the cells remained responsive to calcium for up to 30 min after permeabilization, indicating that they retained their secretory machinery. In the search for a functional role of cytoskeletal proteins in the secretory process, we used SLO-permeabilized cells to examine the localization of filamentous actin, using rhodamine-phalloidin, and that of the actin-severing protein, gelsolin, using specific antibodies. It was found that both F-actin and gelsolin were exclusively localized in the subplasmalemmal region of the cell. We examined the relationship between actin disassembly, the elevation of intracellular calcium and secretion in SLO-treated cells. F-Actin destabilizing agents such as cytochalasin D or DNase I were found to potentiate calcium-stimulated release. The maximal effect was observed at low calcium concentrations (1-4 microM) and at the later stages of the secretory response (after 10 min stimulation). In addition, using rhodamine-phalloidin, we observed that calcium provoked simultaneously both cortical actin disassembly and catecholamine release in SLO-permeabilized cells. These results demonstrate that a close relationship exists between the secretory response and actin disassembly and provide further evidence that intracellular calcium controls the subplasmalemmal cytoskeletal actin organization and thereby the access of secretory granules to exocytotic sites.     

Electrophysiology of a chick neuronal nicotinic acetylcholine receptor expressed in Xenopus oocytes after cDNA injection

Brain nicotinic acetylcholine receptors (nAChRs) are made up of protein subunits that differ from those constituting muscle nAChRs. To characterize the physiological properties of one class of avian brain nicotinic receptor, we injected the nuclei of Xenopus oocytes with full-length cDNAs for the ligand binding (alpha 4) and structural (n alpha) subunits. Injected oocytes had large ACh-induced currents in the microampere range that were insensitive to alpha-bungarotoxin, as expected for neuronal nAChRs. We found that these brain nAChRs incorporate at least two alpha 4 subunits and that their functional properties differ from muscle nAChRs in at least two respects: the elementary conductance is considerably smaller (20 pS), and channels in outside out patches stop functioning within a few minutes.