Notes: Abstract: The myelin P2 protein, a 14,800-Da cytosolic protein found primarily in peripheral nerves, belongs to a family of fatty acid binding proteins. Although it is similar in amino acid sequence and tertiary structure to fatty acid binding proteins found in the liver, adipocytes, and intestine, its expression is limited to the nervous system. It is detected only in myelin-producing cells of the central and peripheral nervous systems, i.e., the oligodendrocytes and Schwann cells, respectively. As part of a program to understand the regulation of expression of this gene, to determine its function in myelin-producing cells, and to study its role in peripheral nerve disease, we have isolated and characterized overlapping human genomic clones encoding the P2 protein. We report here on the partial structure of this gene, and on its localization within the genome. By using a panel of human-hamster somatic cell hybrids and by in situ hybridization, we have mapped the human P2 gene to segment q21 on the long arm of chromosome 8. This result identifies the myelin P2 gene as a candidate gene for autosomal recessive Charcot-Marie-Tooth disease type 4A.

Notes: Abstract: Tyrosine hydroxylase catalyzes the rate-limiting reaction in the biosynthesis of the catecholamine neurotransmitters and hormones (dopamine, norepinephrine, and epinephrine). Rat tyrosine hydroxylase exists, in its native form, as a tetramer composed of identical 498 amino acid subunits. There is currently no information describing the molecular interactions by which the four monomeric tyrosine hydroxylase subunits assemble into an active tetramer. Mutational analysis was performed on bacterially expressed enzyme to assess the role of a putative C-terminal leucine zipper in the assembly of subunits into the tetrameric holoenzyme. Deletion of the C-terminal 19 amino acids, or mutation of a leucine residue (to an alanine), converts the enzyme from a tetrameric to a dimeric form that exhibits greater structural heterogeneity. This change in macromolecular form is accompanied by a 75% (deletion mutation) to 20% (Leu → Ala mutation) reduction in specific activity of the enzyme. This represents the first report of the functional involvement of a region containing a leucine zipper motif in the assembly and activity of a neuronal enzyme.

Notes: Abstract: Estrogens derived from the aromatization of androgens are believed to be responsible for the induction of the sexual differentiation of the CNS interacting with specific estrogen receptors (ER) present in developing neurons. However, the brain cellular distribution of ER is not so well documented. The aim of this study was to investigate the qualitative and quantitative expression of ER mRNA in well characterized cultures of rat type 1 and type 2 astrocytes and of oligodendrocytes by polymerase chain reaction. A series of amplifications with a set of primers spanning along the entire ER mRNA was utilized in the different types of glial cells, in a positive control (uterus), and in a negative control (SK-N-BE cell line) previously shown to be devoid of ER. The data obtained show that ER mRNA is expressed in all three types of glial cell analyzed in almost equal amounts, which are 25–50 times lower than those in the uterus. The mRNA expressed in the glia is homologous with that expressed in the uterine tissue.

Notes: Abstract: As a first step in defining the role of the transforming growth factor-β (TGF-β) superfamily in the development of the sympathetic nervous system, we analyzed effects of several members of this family on neuronal gene expression in dissociated cell culture using a reverse transcription-polymerase chain reaction method. We found that, in addition to activin A, bone morphogenetic protein (BMP)-2 and BMP-6 also induce mRNAs for distinct sets of neuropeptides and neurotransmitter synthetic enzymes in sympathetic neurons. TGF-β1 and TGF-β3 are, however, without detectable effect in this assay. Surprisingly, we find that the patterns of neuropeptide genes induced by activin A, BMP-2, and BMP-6 are each affected differently by neuronal depolarization. Depolarization can either promote or block the effects of different cytokines on the same neuropeptide gene, and depolarization can either promote or block the effects of a given cytokine on different neuropeptide genes. This evidence suggests that neuronal activity may be a key mediator of cytokine modulation of neuronal gene expression.

Notes: Abstract: Effects of ascorbic acid (AA) on 125I-SCH 23982 binding to D1 dopaminergic receptors in membrane preparations from rat striatum were investigated. AA in the range of 0.03 µM–0.33 mM inhibited 75% of specific binding of 125I-SCH 23982 in a dose-dependent manner. At higher concentrations, this inhibition of binding activity by AA was less potent, and 3.3 mM AA inhibited only 30% of specific binding. Reduced glutathione did not alter the inhibition of binding by 0.33 mM AA, but reduced the inhibition by 3.3 mM AA to 8% of specific binding. The loss of specific binding by AA was rescued by 1 mM EDTA, an inhibitor of lipid peroxidation. In the absence of AA, competition experiments with the agonist, dopamine, revealed the presence of high-affinity (Kh = 224.9 ± 48.9 nM) and low-affinity (Kl = 21,100 ± 2,400 nM) binding sites. Although the maximum binding of 125I-SCH 23982 decreased to 40% without affecting the KD value in the presence of 1.67 mM AA, the value of the high-affinity site for dopamine was increased (Kh = 23.3 ± 9.4 nM) and that of the low-affinity site was decreased (Kl = 136,800 ± 40,900 nM). These results suggest that AA may affect D1 dopamine receptor function by lipid peroxidation, competition with dopamine for low-affinity sites, and reduced oxidation of dopamine.

Notes: Abstract: The inhibitory effects of Na+/Ca2+ exchange inhibitory peptide (XIP), which corresponds to residues 219–238 of the Na+/Ca2+ exchange protein from canine heart, were studied in both rat and human brain plasma membrane vesicles. XIP had very high potency with respect to the inhibition of the initial velocity of intravesicular Na+-dependent Ca2+ uptake in both rat brain [IC50 = 3.05 ± 0.69 µM (mean ± SE)] and human brain (IC50 = 3.58 ± 0.58 µM). The maximal inhibition seen in rat brain vesicles was ∼80%, whereas human brain vesicles were inhibited 100%. XIP also inhibited extravesicular Na+-dependent Ca2+ release, and the inhibitory effect was enhanced by increasing the extravesicular Na+ concentration. In contrast, the inhibitory effect of bepridil was competitive with respect to extravesicular Na+. When XIP was added at steady state (5 min after the initiation of intravesicular Na+-dependent Ca2+ uptake), it was found that the intravesicular Ca2+ content declined with time. Analysis of unidirectional fluxes for Ca2+ at steady state showed that 50 µM XIP inhibited Ca2+ influx and efflux ∼85 and 70%, respectively. This result suggested that XIP inhibited both Na+/Ca2+ exchange and Ca2+/Ca2+ exchange but had no effect on the passive release pathway for Ca2+. The results suggest structural homology among cardiac, rat, and human brain exchangers in the XIP binding domain and that the binding of Na+ or other monovalent cations, e.g., K+, is required for XIP to have its inhibitory effect on Ca2+ transport.

Notes: Abstract: In vivo brain microdialysis experiments were performed in the gerbil to evaluate the origin of accumulation of extracellular glutamate under transient ischemia. Microdialysis probes were positioned in the CA1 field of the hippocampus in which proliferation of astrocytes, death of CA1 pyramidal neurons, and damage of presynaptic terminals had been induced by 5-min ischemia 10–14 days before the microdialysis experiment; in the white matter of the cerebral cortex, which contained few neurons, few presynaptic terminals, and many astrocytes; or in the histologically normal CA1 field of the hippocampus, and then 5- or 20-min ischemia was induced. When 5-min ischemia was induced, no significant increase in glutamate content was observed in the CA1 field that showed proliferation of astrocytes, death of CA1 pyramidal neurons, and damage of presynaptic terminals and in the white matter of the cerebral cortex, whereas a significant increase in glutamate (15-fold) was observed in the histologically normal CA1 field. When 20-min ischemia was induced, no significant increase in glutamate content was observed in the CA1 field that showed proliferation of astrocytes, death of CA1 pyramidal neurons, and damage of presynaptic terminals and in the white matter during the first 10 min after the onset of 20-min ischemia, but remarkable ischemia-induced increases in glutamate were observed during the last 10 min of 20-min ischemia in both areas. An excessive increase in glutamate (100-fold) was observed during 20-min ischemia in the normal CA1 field of the hippocampus. When a probe was positioned in the CA1 field of the hippocampus in which presynaptic terminals of Schaffer collaterals and commissural fibers had been eliminated by bilateral kainate injections into the lateral ventricles 4–7 days before the microdialysis experiment and then 5-min ischemia was induced, a significant increase in glutamate was observed during the last half of 5-min ischemia. These results suggest that the efflux of glutamate from astrocytes does not contribute to the large ischemia-induced glutamate accumulation in the CA1 field of the hippocampus during 5-min ischemia but contributes to the ischemia-induced increase in glutamate level during ischemia with a longer duration and that ischemia-induced efflux of glutamate in the CA1 field during 5-min ischemia originates mainly from neuronal elements: presynaptic terminals and postsynaptic neurons.

Notes: Abstract: Paralytic tremor (pt) is a sex-linked mutation in rabbit that affects myelination of the CNS. Myelin in the pt brains represents ∼30% of the normal levels. Previously we showed that the pt mutation affects primarily proteolipid protein (Plp) gene expression. In the present study we investigated the relative effect of the pt mutation on two distinctive Plp gene products, PLP- and DM-20-specific messenger RNAs. Our results showed that both PLP and DM-20 are affected and that the ratio DM-20/PLP was higher in pt rabbits than in age-matched controls. We sequenced normal rabbit PLP cDNA and characterized pt mutation at the DNA level. Rabbit PLP sequence, deduced from cDNA, differs from the human protein only at Thr198. Sequence analysis of the mutant cDNA revealed a transversion T → A in exon 2 of the Plp gene. This point mutation, which is placed at the end of the first potential transmembrane domain, results in a substitution of His36 by a glutamine. This transversion abolishes a restriction site that enabled us to screen a large number of animals and observe a perfect correlation between the pt allele and the abnormal phenotype.

Notes: Abstract: A defect in energy metabolism may play a role in the pathogenesis of neurodegenerative diseases, such as Alzheimer's disease. In the present study, we examined the activities of the enzymes that catalyze oxidative phosphorylation in frontal, temporal, parietal, and occipital cortex from Alzheimer's disease patients and age-matched controls. Complex I and complex II–III activities showed a small decrease in occipital cortex, but were unaffected in the other cortical areas. The most consistent change was a significant decrease of cytochrome oxidase (complex IV) activity of 25–30% in the four cortical regions examined. These results provide further evidence of a cytochrome oxidase defect in Alzheimer's disease postmortem brain tissue. A deficiency in this key energy-metabolizing enzyme could lead to a reduction in energy stores and thereby contribute to the neurodegenerative process.

Notes: Abstract: We have studied the regional distribution and characteristics of polyamine-sensitive [3H]ifenprodil binding sites by quantitative autoradiography in the rat brain. In forebrain areas ifenprodil displaced [3H]ifenprodil (40 nM) in a biphasic manner with IC50 values ranging from 42 to 352 nM and 401 to 974 µM. In hindbrain regions, including the cerebellum, ifenprodil displacement curves were monophasic with IC50 values in the high micromolar range. Wiping studies using forebrain slices (containing both high- and low-affinity sites) or cerebellar slices (containing only the low-affinity site) showed that high- and low-affinity ifenprodil sites are sensitive to spermine and spermidine, to the aminoglycoside antibiotics neomycin, gentamicin, and kanamycin, and to zinc. Two calmodulin antagonists, W7 and calmidazolium, also displaced [3H]ifenprodil from both sites. Other calmodulin antagonists, including trifluoperazine, prenylamine, and chlorpromazine, selectively displaced [3H]ifenprodil from its low-affinity site in hindbrain and forebrain regions. High-affinity [3H]ifenprodil sites, defined either by ifenprodil displacement curves or by [3H]ifenprodil binding in the presence of 1 mM trifluoperazine, were concentrated in the cortex, hippocampus, striatum, and thalamus with little or no labeling of hindbrain or cerebellar regions. This distribution matches that of NMDAR2B mRNA, supporting data showing that ifenprodil has a preferential action at NMDA receptors containing this subunit. Low-affinity [3H]ifenprodil sites have a more ubiquitous distribution but are especially concentrated in the molecular layer of the cerebellum. [3H]Ifenprodil was found to bind to calmodulin-agarose with very low affinity (IC50 of ifenprodil = 516 µM). This binding was displaced by calmodulin antagonists and by polyamines, with a potency that matched their displacement of [3H]ifenprodil from its low-affinity site in brain sections. However, the localization of the low-affinity [3H]ifenprodil site does not strictly correspond to that of calmodulin, and its identity remains to be further characterized. The restricted localization of high-affinity [3H]ifenprodil binding sites to regions rich in NMDAR2B subunit mRNA may explain the atypical nature of this NMDA antagonist.