Quantitation of extrastriatal D2 receptors using a very high-affinity ligand (FLB 457) and the multi-injection approach.
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The multi-injection approach has been used to study in baboon the in vivo interactions between the D2 receptor sites and FLB 457, a ligand with a very high affinity for these receptors. The model structure was composed of four compartments (plasma, free ligand, and specifically and unspecifically bound ligands) and seven parameters (including the D2 receptor site density). The arterial plasma concentration, after correction for metabolites, was used as the input function. The experimental protocol, which consisted of three injections of labeled and/or unlabeled ligand, allowed the evaluation of all model parameters from a single positron emission tomography experiment. In particular, the concentration of receptor sites available for binding (B'max) and the apparent in vivo FLB 457 affinity were estimated in seven brain regions, including the cerebellum and several cortex regions, in which these parameters are estimated in vivo for the first time (B'max is estimated to be 4.0+/-1.3 pmol/mL in the thalamus and from 0.32 to 1.90 pmol/mL in the cortex). A low receptor density was found in the cerebellum (B'max = 0.39+/-0.17 pmol/mL), whereas the cerebellum is usually used as a reference region assumed to be devoid of D2 receptor sites. In spite of this very small concentration (1% of the striatal concentration), and because of the high affinity of the ligand, we demonstrated that after a tracer injection, most of the PET-measured radioactivity in the cerebellum results from the labeled ligand bound to receptor sites. The estimation of all the model parameters allowed simulations that led to a precise knowledge of the FLB 457 kinetics in all brain regions and gave the possibility of testing the equilibrium hypotheses and estimating the biases introduced by the usual simplified approaches. (+info)
Hippocampal EEG and unit activity responses to modulation of serotonergic median raphe neurons in the freely behaving rat.
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Hippocampal EEG, GABAergic interneurons, and principal cells were recorded simultaneously as rats foraged within one of three environments both before and after modulation of serotonergic inputs to the hippocampus. Median raphe microinjections of the 5-HT1a receptor agonist 8-OH-DPAT were made to produce inhibition of serotonergic neurons in this region. Such microinjections produced behavioral arousal and increases in the amplitude of hippocampal EEG theta. Consistent with the pattern of serotonergic innervation of the hippocampus, the GABAergic interneuron population was affected differentially by the microinjections. Principal cells were generally unaffected by the manipulation and maintained robust spatial firing correlates within the foraging environment. The results provide basic data on the relationship between serotonergic median raphe neurons and hippocampal activity in a behaving animal. The data suggest that behavioral responses to manipulation of the serotonergic system are mediated by brain regions other than the hippocampus or are mediated through changes in the activity of hippocampal interneurons. (+info)
Characterization of Xenopus RalB and its involvement in F-actin control during early development.
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We describe the characterization and a functional analysis in Xenopus development of RalB, a small G protein. RalB RNA and protein are detectable during oogenesis and early development, but the gene is expressed only weakly in adult tissues. The RalB transcripts are processed by poly(A) extension during oocyte maturation and up to the gastrulation stage. Microinjection of wild-type or mutant RalB RNAs was performed in fertilized eggs in order to gain insight into the function of RalB during development. We show that during cleavage stages the activated GTP form of RalB specifically induces a cortical reaction that affects the localization of pigment granules. The use of different drugs suggests that this reaction is dependent on the outer cortical actin array. The relation between F-actin and RalB was shown by confocal analysis. Injection of mRNAs encoding the mutated activated form of RalB leads, at dependent doses, to a blocking of gastrulation or defects in closing of neural folding structures. In contrast, the inactivated form blocks only the closing of neural tube. Altogether, these observations suggest that RalB is part of a regulatory pathway that may affect the blastomere cytoskeleton and take part in early development. (+info)
Evidence for the involvement of a Src-related tyrosine kinase in Xenopus egg activation.
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Recently, we have purified a Src-related tyrosine kinase, named Xenopus tyrosine kinase (Xyk), from oocytes of Xenopus laevis and found that the enzyme is activated within 1 min following fertilization [Sato et al. (1996) J. Biol. Chem. 271, 13250-13257]. A concomitant translocation of a part of the activated enzyme from the membrane fraction to the cytosolic fraction was also observed. In the present study, we show that parthenogenetic egg activation by a synthetic RGDS peptide [Y. Iwao and T. Fujimura, T. (1996) Dev. Biol. 177, 558-567], an integrin-interacting peptide, but not by electrical shock or the calcium ionophore A23187 causes the kinase activation, tyrosine phosphorylation, and translocation of Xyk. A synthetic tyrosine kinase-specific inhibitor peptide was employed to analyze the importance of the Xyk activity in egg activation. We found that the peptide inhibits the kinase activity of purified Xyk at IC50 of 8 microM. Further, egg activation induced by sperm or RGDS peptide but not by A23187 was inhibited by microinjection of the peptide. In the peptide-microinjected eggs, penetration of the sperm nucleus into the egg cytoplasm and meiotic resumption in the egg were blocked. Indirect immunofluorescence study demonstrates that Xyk is exclusively localized to the cortex of Xenopus eggs, indicating that Xyk can function in close proximity to the sperm-egg or RGDS peptide-egg interaction site. Taken together, these data suggest that the tyrosine kinase Xyk plays an important role in the early events of Xenopus egg activation in a manner independent or upstream of calcium signaling. (+info)
Nr-CAM promotes neurite outgrowth from peripheral ganglia by a mechanism involving axonin-1 as a neuronal receptor.
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Nr-CAM is a neuronal cell adhesion molecule (CAM) belonging to the immunoglobulin superfamily that has been implicated as a ligand for another CAM, axonin-1, in guidance of commissural axons across the floor plate in the spinal cord. Nr-CAM also serves as a neuronal receptor for several other cell surface molecules, but its role as a ligand in neurite outgrowth is poorly understood. We studied this problem using a chimeric Fc-fusion protein of the extracellular region of Nr-CAM (Nr-Fc) and investigated potential neuronal receptors in the developing peripheral nervous system. A recombinant Nr-CAM-Fc fusion protein, containing all six Ig domains and the first two fibronectin type III repeats of the extracellular region of Nr-CAM, retains cellular and molecular binding activities of the native protein. Injection of Nr-Fc into the central canal of the developing chick spinal cord in ovo resulted in guidance errors for commissural axons in the vicinity of the floor plate. This effect is similar to that resulting from treatment with antibodies against axonin-1, confirming that axonin-1/Nr-CAM interactions are important for guidance of commissural axons through a spatially and temporally restricted Nr-CAM positive domain in the ventral spinal cord. When tested as a substrate, Nr-Fc induced robust neurite outgrowth from dorsal root ganglion and sympathetic ganglion neurons, but it was not effective for tectal and forebrain neurons. The peripheral but not the central neurons expressed high levels of axonin-1 both in vitro and in vivo. Moreover, antibodies against axonin-1 inhibited Nr-Fc-induced neurite outgrowth, indicating that axonin-1 is a neuronal receptor for Nr-CAM on these peripheral ganglion neurons. The results demonstrate a role for Nr-CAM as a ligand in axon growth by a mechanism involving axonin-1 as a neuronal receptor and suggest that dynamic changes in Nr-CAM expression can modulate axonal growth and guidance during development. (+info)
SNAP23 promotes insulin-dependent glucose uptake in 3T3-L1 adipocytes: possible interaction with cytoskeleton.
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The acute stimulation of glucose uptake by insulin in fat and muscle cells is primarily the result of translocation of facilitative glucose transporter 4 (GLUT-4) from an internal compartment to the plasma membrane. Here, we investigate the role of SNAP23 (a 23-kDa molecule resembling the 25-kDa synaptosome associated protein) in GLUT-4 translocation and glucose uptake in 3T3-L1 adipocytes. Microinjection of a polyclonal antibody directed to the carboxy terminus of SNAP23 inhibited GLUT-4 incorporation into the membrane in response to insulin, whereas microinjection of full-length recombinant SNAP23 enhanced the insulin effect. Introduction of recombinant SNAP23 into chemically permeabilized cells also enhanced insulin-stimulated glucose transport. These results indicate that SNAP23 is required for insulin-dependent, functional incorporation of GLUT-4 into the plasma membrane and that the carboxy terminus of the protein is essential for this process. SNAP23 is therefore likely to be a fusion catalyst along with syntaxin-4 and vesicle-associated membrane protein (VAMP)-2. Furthermore, the endogenous content of SNAP23 appears to be limiting for insulin-dependent GLUT-4 exposure at the cell surface. A measurable fraction of SNAP23 was sedimented with cytoskeletal elements when extracted with Triton X-100, unlike VAMP-2 and syntaxin-4, which were exclusively soluble in detergent. We hypothesize that SNAP23 and its interaction with the cytoskeleton may be targets for regulation of GLUT-4 traffic. (+info)
Calcium release and subsequent development induced by modification of sulfhydryl groups in porcine oocytes.
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The mechanism of Ca2+ release induced by modification of sulfhydryl groups and the subsequent activation of porcine oocytes were investigated. Thimerosal, a sulfhydryl-oxidizing compound, induced Ca2+ oscillation in matured oocytes. In thimerosal-preincubated oocytes, the amount of Ca2+ released after microinjection of inositol 1,4,5-trisphosphate (InsP3) or ryanodine increased strikingly, indicating that thimerosal potentiated both InsP3- and ryanodine-sensitive Ca2+ release pathways. Thimerosal also enhanced the sensitivity of oocytes to microinjected Ca2+ so that in pretreated oocytes a Ca2+ injection triggered a larger transient. Heparin at concentrations that normally block the InsP3-induced Ca2+ release were without effect; higher doses significantly increased the time leading up to the first spike. The thimerosal-induced Ca2+ release could not be blocked by procaine, and it did not require the formation of InsP3 since preinjection with neomycin did not prevent the oscillation. Immunocytochemistry revealed that thimerosal treatment destroyed the meiotic spindle, preventing further development, an effect that could be reversed by dithiothreitol. The combined thimerosal/dithiothreitol treatment triggered second polar body extrusion in 50% of the oocytes, and as a result of this activation scheme approximately 15% of the in vitro- and approximately 60% of the in vivo-matured oocytes developed to blastocyst during a 7-day culture in vitro. (+info)
Ajuba, a novel LIM protein, interacts with Grb2, augments mitogen-activated protein kinase activity in fibroblasts, and promotes meiotic maturation of Xenopus oocytes in a Grb2- and Ras-dependent manner.
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LIM domain-containing proteins contribute to cell fate determination, the regulation of cell proliferation and differentiation, and remodeling of the cell cytoskeleton. These proteins can be found in the cell nucleus, cytoplasm, or both. Whether and how cytoplasmic LIM proteins contribute to the cellular response to extracellular stimuli is an area of active investigation. We have identified and characterized a new LIM protein, Ajuba. Although predominantly a cytosolic protein, in contrast to other like proteins, it did not localize to sites of cellular adhesion to extracellular matrix or interact with the actin cytoskeleton. Removal of the pre-LIM domain of Ajuba, including a putative nuclear export signal, led to an accumulation of the LIM domains in the cell nucleus. The pre-LIM domain contains two putative proline-rich SH3 recognition motifs. Ajuba specifically associated with Grb2 in vitro and in vivo. The interaction between these proteins was mediated by either SH3 domain of Grb2 and the N-terminal proline-rich pre-LIM domain of Ajuba. In fibroblasts expressing Ajuba mitogen-activated protein kinase activity persisted despite serum starvation and upon serum stimulation generated levels fivefold higher than that seen in control cells. Finally, when Ajuba was expressed in fully developed Xenopus oocytes, it promoted meiotic maturation in a Grb2- and Ras-dependent manner. (+info)