Neuroprotocols最新文献

The study of axon growth in culture is limited by a poor understanding of the relative contribution of each of a complex array of factors, which include diffusible, axon growth-modulating molecules and substrate-bound guidance cues available to developing and regenerating neurons in vivo. With the objective of more closely mimicking in vivo conditions, one approach we have exploited employs thin cryosections of appropriate regions of unfixed nervous tissue as culture substrata for the growth of regenerating neurons. By using this technique it is possible to culture different populations of neurons on substrata in which environmental growth-modulating factors are preserved. This form of bioassay has facilitated the study of the different neurite outgrowth responses of neurons both from different sources and at different developmental ages on varying native substrata. Using this method we have demonstrated that mature dorsal root ganglion neurons (DRG) will regrow axons only on predegenerated sciatic nerve in vitro, while immature DRG extend neurites on both intact and degenerated sciatic nerve. In contrast, both mature and neonatal DRG fail to regenerate on either fully myelinated mature optic nerve or unmyelinated embryonic optic nerve. Moreover, neonatal retinal ganglion cells do not regenerate on any of these substrata.

The reaction of growth cones in in vitro assays to substrate-bound molecules might yield important clues to the roles that these molecules play in growth cone guidance in vivo. Janusin and tenascin are glia-derived, extracellular matrix molecules that are expressed in the nervous system at times and in locations that suggest that they might act as barriers to neurite outgrowth. To test this hypothesis we have used video time-lapse microscopy to observe the behavior of growth cones, growing on a substrate permissive for neurite outgrowth, when they are confronted with janusin or tenascin as sharp, substrate boundaries. Here we describe the method for offering growth cones a choice between two substrates, in which the border between the two molecules can be clearly visualized in the phase-contrast microscope during the period of observation. We have learned from these observations that growth cones avoid advancing onto janusin or tenascin substrates, but do not undergo gross morphological changes, such as complete collapse, when they contact these molecules.

The adrenergic receptors belong to a family of receptors that is postulated to span the plasma membrane seven times and is linked to regulatory GTP-binding proteins. These receptors mediate a wide variety of physiological responses through activation of several distinct second-messenger systems. Belying the importance of the adrenergic receptors in regulating physiological responses in target cells is their paucity. These receptors are present in very low numbers, in some cases only a thousand copies per cell. In addition, the adrenergic receptors are relatively stable proteins. Thus, cells have no need to synthesize large amounts of these proteins and, in consequence, the level of mRNA coding for these receptors is very low. In this paper, I examine some of the ways in which quantitation of these scarce mRNA species has been approached, with particular emphasis on the use of polymerase chain reaction.

The successful synthesis and use of carrier-free radioiodinated β₂-adrenergic receptor competitive antagonist photoaffinity labels (±)-[¹²⁵I]IABP, (±)-[¹²⁵I]MAPIT, (−)-[¹²⁵I]IAPTA, and (±)-[¹²⁵I]IAPCGP-12177, are described. In addition, the synthesis and use of two carrier-free radioiodinated β-adrenergic receptor agonist photoaffinity labels (±)-[¹²⁵I]iodoazidoprenalterol ((±)-[¹²⁵I]IAPr) and (−)-N-(p-azido-m-[¹²⁵I]iodophenethylamidoisobutyl)norepinephrine ((−)-[¹²⁵I]NAIN), are described. All antagonist photolabels were capable of highly specific derivatization of the purified recombinant hamster lung β₂-adrenergic receptor. Tryptic cleavage of the photolabeled receptor into a 30-kDa radiolabeled fragment (transmembrane 1-5) and an 8-kDa radiolabeled fragment (transmembrane 6,7) showed variable Insertion ratios between the two juxtaposed domains, depending on the structure of the photolabel. Unique synthetic strategies were used for the agonist photolabels. The phenolic hydroxyl of (±)-IAPr was protected as the glucoside and deprotected enzymatically in the final step. The final coupling step in the synthesis of (−)-[¹²⁵I]NAIN was accomplished by reductive alkylation without protection of the catechol hydroxyls of norepinephrine using sodium cyanoborohydride. (±)-IAPr was found to be a partial agonist for the turkey erythrocyte β-adrenergic receptor and an effective photoaffinity label for the avian β-adrenergic receptor. (−)-NAIN was found to be a full agonist for the β₂-adrenergic receptor in guinea pig lung membranes and a highly effective agonist photoaffinity label for the β₂-adrenergic receptor. These photolabels will be useful for probing the β-adrenergic receptor binding site In order to "map" this site under nonactivated (antagonist photolabels) or activated states (agonist photolabels).

The application of in vitro site-directed mutagenesis has led to the identification of conserved amino acids that play important roles in receptor structure and function. Precise amino acid substitutions can be obtained and then correlated with changes in receptor phenotype. Here, we describe several techniques commonly employed to Introduce site-specific mutations. The benefits and potential drawbacks of each method are discussed. Site-directed mutagenesis of the human α_2A-adrenergic receptor (α_2AAR) has been successfully employed to identify conserved amino acids involved in agonist binding and receptor activation. Aspartate residues in the second and intracellular side of the third transmembrane domain of the α_2AAR are implicated in receptor/G-protein interactions. Since these aspartate residues are highly conserved among all G-protein-coupled receptors, and elimination of these residues has been shown to abolish the ability of other receptors in this class to activate their respective intracellular signaling pathways, It seems likely that these residues are critical for agonist-induced conformational changes that underlie receptor/G-protein interactions. In contrast to the role played by the conserved residues mentioned above, a conserved aspartate residue situated near the extracellular side of the third transmembrane domain plays a pivotal role in adrenergic ligand binding. Genetic analysis of the fifth transmembrane domain of the α_2AAR suggests that a conserved serine residue in this region participates in hydrogen binding to the meta-hydroxyl group of catecholamines. These findings point to the utility of site-directed mutagenesis in identifying structure-function relationships among G-protein-coupled receptors.