Ontogenetic and Imprinting-Induced Changes in Chick Brain Protein Metabolism and Muscarinic Receptor Binding Activity

Abstract— Over the 20-min period following exposure of young chicks to a flashing light as an imprinting stimulus there is an increased incorporation of [¹⁴C]leucine into an acidic (tubulin-enriched) protein fraction of the anterior dorsal forebrain in birds which have learnt the characteristics of the stimulus as compared with, either birds which have been exposed to an imprinting stimulus but learn poorly, or chicks kept in the dark. This brain region has been implicated in several studies as the locus for a number of biochemical modulations that accompany learning. The amount of [¹⁴C]leucine incorporated does not seem to be determined by precursor pool availability; it does, however, correlate with a well-validated measure of the extent to which birds have learnt to recognise the characteristics of the stimulus, as shown by a two-choice discrimination test. There is no change in the total content of tubulin dimer as assayed by colchicine binding under these conditions. Additionally, in birds which show evidence of learning, the binding of quinuclidinyl benzilate, an irreversible muscarinic ligand, is altered in both the posterior dorsal forebrain and midbrain regions. None of these effects could be simply the result of visual stimulation. The meaning of these changes is discussed.     

Developmental Expression of Transferrin Binding Protein in Oligodendrocyte Lineage Cells of the Embryonic Chick Spinal Cord

Oligodendrocytes develop from precursor cells in the neuroepithelium of the ventral ventricular zone. Oligodendrocytes in the different stages of development are characterized by expression of a number of different marker molecules such as myelin genes, growth factors, and specific antigens. We have previously identified that transferrin binding protein (TfBP), a member of heat shock protein 90 families, is a novel avian ER-associated membrane protein that is specifically localized in oligodendrocytes in adult chicken CNS. In this study we describe the developmental expression of TfBP in the embryonic chick spinal cord. A few, distinct, TfBP+ cells appeared at the lateral margin of the subventricular neuroepithelium of the spinal cord at E7. Thereafter, some TfBP+ cells, exhibited a migrative form of unipolar or bipolar shape occurred around E8 in the mantle layer, midway between the neuroepithelium and the marginal layer of the primitive spinal cord. Thereafter, the TfBP+ cells rapidly increased in number as well as their staining intensity, and overall distribution of TfBP+ cells at E15 was comparable to that of a mature spinal cord. Our observations suggest that TfBP is expressed in the subpopulation of oligodednrcyte lineage in the development and a putative role of TfBP in relation to transferrin and iron trafficking is considered. SW Park and HS Lim contributed equally to this work.     

Characterization of Myelin-Associated Glycoprotein (MAG) Proteolysis in the Human Central Nervous System

Purified human central nervous system myelin contains an endogenous cysteine protease which degrades the 100-kDa myelin-associated glycoprotein into a slightly smaller 90-kDa derivative called dMAG, and which has been implicated in demyelinating diseases. The native proteolytic site in human MAG was determined in order to characterize this cysteine protease in humans further. This was accomplished by identifying the carboxy-terminus of purified dMAG. The results of these experiments, in conjunction with peptidolysis assays of myelin, demonstrated that the enzyme which proteolyses MAG is extracellular and has cathepsin L-like specificity. Furthermore, it was shown that this cathepsin L-like activity potentially was regulated by the endogenous extracellular inhibitor cystatin C.     

记忆增强肽促进大鼠海马内CREB磷酸化

五肽ＺＮＣ（ＣＰＲ（ｐＢＧｌｕ－Ａｓｎ－Ｃｙｔ－Ｐｒｏ－Ａｒｇ－ＯＨ）是精氨酸加压素（ＡＶＰ）在脑内的天然酶解产物,具有促进学习记忆的中枢效应。为了进一步阐明其作用的分子机制,以整体大鼠海马及离体大鼠海马切片为对象,研究了ＺＮＣ（Ｃ）ＰＲ对ｃＡＭＰ反应元件结合蛋白（ＣＲＥＢ）磷酸化的作用。发现ＺＮＣ（Ｃ）ＰＲ及其类似物ＮＬＰＲ能诱导大鼠海马内ＣＲＥＢ磷酸２化,该作用能被其拮抗剂ＺＤＣ（Ｃ）ＰＲ、Ｇ     

Inhibition of the Chick Pineal Rhythm in Rate of Thymidine Incorporation by Cyclic AMP

Chick pineal glands in organ culture showed a circadian rhythm in the rate of thymidine incorporation. Thymidine incorporation was very markedly inhibited when 3-isobutyl-l-methylxanthine (IBMX) was continuously present. When IBMX was added to cultures in control medium during the photoperiod of the second day in culture, the extent of inhibition of incorporation during that photoperiod increased with the increase in length of the photoperiod remaining. Incorporation did not resume at the start of a second photoperiod if IBMX was added within the first 10 h of the first photoperiod. Corresponding results were obtained with glands continuously cultured in constant darkness. Similar results were also obtained using glands treated with 4-(3-butoxy-4-methoxybenzyl)-2-imidazolidinone (Ro 20-1724), 7 beta-acetoxy-8,13-epoxy-1 alpha, 6 beta, 9 alpha-trihydroxy-1abd-14-ene-11-one (forskolin), or 8-bromo-cyclic AMP, but not with 8-bromo-cyclic GMP. When glands cultured with IBMX were transferred to control medium, incorporation remained inhibited until the start of the next photoperiod. We conclude that the increase in the rate of thymidine incorporation at the start of each new photoperiod is dependent on a "switch" process that is inhibited by elevated concentrations of cyclic AMP.     

Chromodomain Helicase Binding Protein 8 (Chd8) Is a Novel A-Kinase Anchoring Protein Expressed during Rat Cardiac Development

A-kinase anchoring proteins (AKAPs) bind the regulatory subunits of protein kinase A (PKA) and localize the holoenzyme to discrete signaling microdomains in multiple subcellular compartments. Despite emerging evidence for a nuclear pool of PKA that rapidly responds to activation of the PKA signaling cascade, only a few AKAPs have been identified that localize to the nucleus. Here we show a PKA-binding domain in the amino terminus of Chd8, and demonstrate subcellular colocalization of Chd8 with RII. RII overlay and immunoprecipitation assays demonstrate binding between Chd8-S and RIIα. Binding is abrogated upon dephosphorylation of RIIα. By immunofluorescence, we identified nuclear and perinuclear pools of Chd8 in HeLa cells and rat neonatal cardiomyocytes. We also show high levels of Chd8 mRNA in RNA extracted from post-natal rat hearts. These data add Chd8 to the short list of known nuclear AKAPs, and implicate a function for Chd8 in post-natal rat cardiac development.     

Astrocytes and Interneurons in Memory Processing in the Chick Hippocampus: Roles for G-Coupled Protein Receptors,GABA(B) and mGluR1

Glutamate and GABA acting at mGluR1 and GABA_B receptors, respectively, have roles in memory processing in the hippocampus up to 35 min after bead discrimination learning in the young chick. Activation of mGluR1 receptors is important at 2.5 and 30 min after training, but modulation of these receptors between these two times has no effect on memory. This timing is similar to the action of glutamate on NMDA receptors. The GABA_B antagonist, phaclofen, and the inhibitor of astrocytic oxidative metabolism, fluoroacetate, inhibited memory when injected between 2.5 and 30 min. Paradoxically, a high dose of the GABA_B agonist, baclofen, also inhibited memory, but a low dose promoted memory consolidation—an effect possibly caused by too much information and loss of the ‘message’. These results are interpreted in terms interactions between interneurons, astrocytes and pyramidal cells and demonstrate the importance of all cell types in memory processing in the hippocampus.     

Suppressive effect of camostat mesilate (FOY 305) on acute experimental allergic encephalomyelitis (EAE)

Camostat mesilate (FOY305), a synthetic serine protease inhibitor and has been developed as a crug for pancreatitis, is effective in suppressing acute experimental allergic encephalomyelitis in Lewis rats. Loss of weight, clinical score and yield of myelin protein from brain stem were improved by daily injection of FOY305 compared with saline from day 6 after inoculation with homogenate of guinea pig spinal cord. A significant decrease of yield of myelin has been shown here for the first time in acute EAE in Lewis rat. This is in accord with myelin breakdown demonstrated morphologically. Our study also demonstrates a significant improvement of yield of myelin protein by FOY305. Our results suggest the possibility of a clinical application of this protease inhibitor for human demyelinating diseases such as multiple sclerosis.     

Susceptibility of myelin proteins to a neutral endoproteinase: The degradation of myelin basic protein (MBP) and P2 protein by purified bovine brain multicatalytic proteinase complex (MPC)

Multicatalytic proteinase complex (MPC) was isolated from bovine brain and the susceptibility of myelin basic protein (MBP) and P₂ protein of bovine central and peripheral nervous system was examined. SDS-polyacrylamide electrophoretic analysis of purified MPC revealed protein bands of molecular weight ranging from 22–35 kDa. The enzyme is activated by SDS at a concentration less than 0.01%. Upon incubation with MPC, purified MBP and P₂ proteins were degraded into smaller fragments. There was a 57% and 100% loss of MBP at 2 and 6 hours of incubation. The P₂ protein which is not susceptible to any endogenous non-lysosomal enzyme thus far studied was digested into small peptide fragments only in the presence of SDS (0.01%) and not in its absence. These results indicate that MPC which is active at physiological conditions may have a role in the turnover of myelin proteins and in demyelinating diseases.     

Targeted deletion of the antisilencer/enhancer (ASE) element from intron 1 of the myelin proteolipid protein gene (Plp1) in mouse reveals that the element is dispensable for Plp1 expression in brain during development and remyelination

Myelin proteolipid protein gene (Plp1) expression is temporally regulated in brain, which peaks during the active myelination period of CNS development. Previous studies with Plp1‐lacZ transgenic mice demonstrated that (mouse) Plp1 intron 1 DNA is required for high levels of expression in oligodendrocytes. Deletion‐transfection analysis revealed the intron contains a single positive regulatory element operative in the N20.1 oligodendroglial cell line, which was named ASE (a ntis ilencer/e nhancer) based on its functional properties in these cells. To investigate the role of the ASE in vivo, the element was deleted from the native gene in mouse using a Cre/lox strategy. Although removal of the ASE from Plp1‐lacZ constructs profoundly decreased expression in transfected oligodendroglial cell lines (N20.1 and Oli‐neu), the element was dispensable to achieve normal levels of Plp1 gene expression in mouse during development (except perhaps at postnatal day 15) and throughout the remyelination period following cuprizone‐induced (acute) demyelination. Thus, it is possible that the ASE is non‐functional in vivo, or that loss of the ASE from the native gene in mouse can be compensated for by the presence of other regulatory elements within the Plp1 gene.