Kyoto University (京都大学) / 0048 / 新制・課程博士 / 博士(工学) / 甲第15675号 / 工博第3333号 / 新制||工||1503(附属図書館) / 28212 / 京都大学大学院工学研究科合成・生物化学専攻 / (主査)教授 森 泰生, 教授 跡見 晴幸, 教授 濵地 格 / 学位規則第4条第1項該当

The concerns of the present study were to determine 1) the conditions necessary to measure displaceable [³H] L-aspartate binding to membrane fractions of the rat brain, 2) whether the binding demonstrated the charcteristics of the site which is active in vivo, and 3) whether the acidic amino acid neurotransmitters aspartate and glutamate bind to identical or different sites by comparing the pharmacological specificities of the [³H] L-aspartate binding with that of [³H] L-glutamate. The conditions of the [³H] L-aspartate binding assay were determined in synaptosomal and total particulate fractions of whole rat brain. The reaction mixture which included the membrane fraction suspended in Tris-HCl buffer (pH 7.4) in the presence or absence of the compound under test, was incubated at 37°C for 30 minutes. The reaction was stopped by centrifugation and the radioactivity in the pellet counted by liquid scintillation spectrometry. The [³H] L-aspartate binding was characterized in total particulate fractions of rat cerebellum. The apparent dissociation constant (K[sub=D]) and maximum binding (Bmax), as determined by Scatchard analysis, are 1.64 ± 0.34 μM and 7711 ± fmol/mg protein respectively. The displaceable binding is reversible, saturable, independent of the presence of NA⁺, has an affinity in the range where the neurotransmitter is active in vivo, and demonstrates a pharmacological specificity which includes stereospecificity. The compounds tested to demonstrate the pharmacological specificity were L-aspartate (IC[sub=50] = 1.81 μM), D-aspartate (IC[sub=50] = 46.6 μM), L-glutamate (IC[sub=50] = 1.24 μM), N-methyl-DL-aspartate (inactive), kainate (inactive), D-alpha-aminoadipate (inactive), and L-alpha-aminoadipate (IC[sub=50] =7.12 μM). The pharmacological specificity of [³H] L-aspartate binding was different from that of [³H] L-glutamate. When the binding data only are considered, therefore, separate receptors for aspartate and glutamate are indicated. The pharmacological specificity of the [³H] L-aspartate binding, that is the affinity of the binding site for N-methyl-DL-aspartate, D- and L-alpha-aminoadipate, however, does not correlate with the potency of these compounds derived from iontophoretic studies. L-alpha-aminoadipate is very effective while N-methyl-DL-aspartate and D-alpha-aminoadipate do not displace the [³H] L-aspartate binding. In iontophoretic studies, N-methyl-D-aspartate and D-alpha-aminoadipate are very potent as compared to aspartate while L-alpha-aminoadipate Is inactive. The [³H] L-aspartate binding then may not represent the site which is active in vivo. The characteristics of the aspartate site in vivo, however, may not be truely represented in iontophoretic studies because of, for example, uptake of the compounds. The aspartate binding site, therefore, must be identified as that which is activated in vivo. The question of separate receptors for aspartate and glutamate then must still be resolved. / Science, Faculty of / Zoology, Department of / Graduate

Digital averaging and analysis of miniature endplate currents (MEPCs) from mouse diaphragm was used to characterize the normal MEPC and its modification by a variety of drugs. Under normal conditions the decay of MEPCs showed consistent deviations from a simple exponential consisting in a progressive increase of rate constant, followed by a slow tail. Receptor blockade by d-tubocurarine (dTC), a-bungarotoxin, and other agents thought to occupy ACh-binding sites reduced MEPC amplitude, accelerated MEPC decay by about 30% (making it about equal to decay rate of channels opened by exogenous acetylcholine), and eliminated the early deviations from an exponential decay; dTC also abolished the late tail. Examination of the interaction of acetylcholinesterase (AChE) poisoning and receptor blockade on MEPC height and time course indicated that normally most quantal ACh is captured by receptors and, as predicted by theoretical consideration, a rather large degree of receptor blockade is necessary to reduce MEPC height. MEPC tails were exaggerated by AChE poisoning and exogenous ACh or carba-chol. The latter agents reduced MEPC height in a fashion inconsistent with blockade of ACh binding and concurrent modulation of the tail suggested an important role of desensitized receptors in tail generation. A number of other drug actions are also described quantitatively: (a) channel prolongation, typical of alcohols but also found with ketones and some amines; (b) 'channel plugging', typical of local anaesthetics but also found with many other agents, including long chain alcohols, and (c) an action to reduce MEPC size without reducing net response to exogenous agonist typical of volatile anaesthetics, associated with increase rather than decrease of ACh binding to receptor. Criteria for distinguishing different modes of modification of receptor function are discussed. / Medicine, Faculty of / Anesthesiology, Pharmacology and Therapeutics, Department of / Graduate

Anandamide, N-arachidonylethanolamide (NAE 20:4), is an endocannabinoid receptor ligand unique to animals, in which it influences a wide range of physiological and behavioral functions. Using selective lipidomics approach, we recently identified occurrence of anandamide or NAE 20:4 and its precursor in moss plants. While Nacylethanolamines (NAEs) with C12-C18 acyl chain are ubiquitous in seed plants and play a role in mediating abscisic acid (ABA)-dependent or -independent responses to stress, endocannabinoid receptor-mediated interactions, similar to that of animals, have not been elucidated for plants. Physcomitrella patens provides us with a unique opportunity to address if 1) early land plants, such as mosses, retained NAE-mediated signaling mechanism that is akin to animals but not to vascular plants and 2) if such distinctive NAE profile and mechanisms by which it may function in moss plants is responsible, in part, for their natural ability to resist high temperatures and tolerate osmotic and salt stresses and dehydration. Our current studies are focused on characterization of anandamide metabolic pathway and its functional role in the development of moss. Insights into unique lipid composition and signaling pathways that mosses acquired naturally, during their successful transition from water to land, may lead to development of tools necessary to enhance abiotic stress tolerance in other plants.

1. The role of calmodulin (CaM) in transmitter release was investigated using liposomes to deliver CaM and monoclonal antibodies against CaM (antiCaM) directly into the frog motor nerve terminal. 2. Miniature endplate potentials (MEPPs) were recorded in a high K+ solution, and effects on transmitter release were monitored using estimates of the quantal release parameters m (number of quanta released), n (number of functional transmitter release sites), p (mean probability of release), and vars p (spatial variance in p). 3. Administration of CaM, but not heat-inactivated CaM, encapsulated in liposomes (1000 units ml-1) produced an increase in m (25%) that was due to an increase in n. MEPP amplitude was not altered by CaM. 4. Administration of antiCaM, but not heat-inactivated antiCaM, in liposomes (50 μl ml-1) produced a progressive decrease in m (40%) that was associated with decreases in n and p. MEPP amplitude was decreased (15%) after a 25 min lag time, suggesting a separation in time between the decreases in quantal release and quantal size. 5. Bath application of the membrane-permeable CaM antagonist W7 (28 μM) produced a gradual decrease in m (25%) that was associated with a decrease in n. W7 also produced a decrease in MEPP amplitude that paralleled the decrease in m. The decreases in MEPP size and m produced by W7 were both reversed by addition of CaM. 6. Our results suggest that CaM increases transmitter release by mobilizing synaptic vesicles at the frog motor nerve terminal.

In this dissertation I describe the isolation and characterization of genomic clones for the human synapsin I gene, the establishment of a linkage map for the human synapsin I gene locus, and studies of the possible involvement of this gene in neurological disease. Synapsin I is a neuron-specific phosphoprotein which is concentrated at the presynaptic terminal. Evidence suggests that it plays a fundamental role in the regulation of neurotransmitter release. Altogether 27,500 bp of the human synapsin I gene have been isolated, and the gene structure has been partially determined. DNA sequence comparisons between human and rat genes show a high degree of conservation. Sequenced exons display an 87% identity to each other. The synapsin I genomic clones were employed in the search for a polymorphic marker. A compound (AC)n repeat located 1000 base pairs downstream from the human synapsin I gene and within the last intron of the A-raf-1 gene has been identified. DNA database comparisons of the sequences surrounding the repeat indicate that the synapsin I gene and the A-raf-1 gene lie immediately adjacent to each other, in opposite orientation. Polymerase chain reaction amplification of this synapsin I / A-raf-1 associated repeat using total genomic DNA from members of the 40 reference pedigree families of the Centre d'Etude du Polymorphisme Humaine showed it to be highly polymorphic, with a polymorphic information content value of 0.84 and a minimum of eight alleles. Because the synapsin I gene had been mapped previously to the short arm of the human X chromosome at Xp11.2, linkage analysis was performed with markers on the proximal short arm of the X chromosome. The most likely gene order is: DXS7 - SYN/ARAF1 - TIMP - DXS255 - DXS146 with a relative probability of 5 x 108 compared with the next most likely order. The SynI/Araf marker was next utilized in a linkage study aimed at establishing a more accurate placement of the genetic locus responsible for the ocular disorder Congenital stationary night blindness, which had been mapped previously close to DXS7. Our results confirm this prior localization and also exclude any placement proximal to the SYN/ARAF1 locus. Finally, the inheritance of the different alleles of the SynI/Araf marker in three families with Rett syndrome, a severe neurodegenerative disorder, which has been assigned to the X chromosome, was studied. In at least one of the families in which two half sisters with the same mother suffer from the disease, the inheritance of Rett syndrome was discordant with the inheritance of the same allele for the SynI/Araf marker. Thus, this highly informative repeat has proven already effective in the study of X-linked diseases and should serve as a valuable marker for disease loci mapped to the Xp11 region.

No description available.

京都大学 / 0048 / 新制・課程博士 / 博士(工学) / 甲第18594号 / 工博第3955号 / 新制||工||1608(附属図書館) / 31494 / 京都大学大学院工学研究科合成・生物化学専攻 / (主査)教授 森 泰生, 教授 梅田 眞郷, 教授 濵地 格 / 学位規則第4条第1項該当 / Doctor of Philosophy (Engineering) / Kyoto University / DGAM

京都大学 / 0048 / 新制・課程博士 / 博士(工学) / 甲第19376号 / 工博第4121号 / 新制||工||1635(附属図書館) / 32390 / 新制||工||1635 / 京都大学大学院工学研究科合成・生物化学専攻 / (主査)教授 森 泰生, 教授 梅田 眞郷, 教授 濵地 格 / 学位規則第4条第1項該当 / Doctor of Philosophy (Engineering) / Kyoto University / DFAM

Indiana University-Purdue University Indianapolis (IUPUI) / Voltage-gated sodium channels (VGSCs) are dynamic membrane spanning proteins which mediate the rapid influx of Na+ during the upstroke of the action potential (AP). In addition to the large inward Na+ currents responsible for the upstroke of the AP, some VGSC isoforms produce smaller, subthreshold Na+ currents, which can influence the excitable properties of neurons. An example of such a subthreshold current is resurgent Na+ current (INaR). These unusual currents are active during repolarization of the membrane potential, where the channel is normally refractory to activity. INaR exhibit slow gating kinetics and unusual voltage-dependence derived from a novel mechanism of channel inactivation which allows the channel to recover through an open configuration resulting in membrane depolarization early in the falling phase of the AP, ultra-fast re-priming of channels, and multiple AP spikes. Although originally identified in fast spiking central nervous system (CNS) neurons, INaR has recently been observed in a subpopulation of peripheral dorsal root ganglion (DRG) neurons. Because INaR is believed to contribute to spontaneous and high frequency firing of APs, I have hypothesized that increased INaR may contribute to ectopic AP firing associated with inherited and acquired disorders of excitability. Specifically, this dissertation explores the mechanisms which underlie the electrogenesis of INaR in DRG neurons and determines whether the biophysical properties of these unique currents were altered by mutations that cause inherited muscle and neuronal channelopathies or in an experimental model of nerve injury. The results demonstrate that (1) multiple Na+ channel isoforms are capable of producing INaR in DRG neurons, including NaV1.3, NaV1.6, and NaV1.7, (2) inherited muscle and neuronal channelopathIy mutations that slow the rate of channel inactivation increase INaR amplitude, (3) temperature sensitive INaR produced by select skeletal muscle channelopthy mutations may contribute to the triggering of cold-induced myotonia, and (4) INaR amplitude and distribution is significantly increased two weeks post contusive spinal cord injury (SCI). Taken together, results from this dissertation provide foundational knowledge of the properties and mechanism of INaR in DRG neurons and indicates that increased INaR likely contributes to the enhanced membrane excitability associated with multiple inherited and acquired disorders of excitability.