Keiko Hirai

Functional correlation of fetal and adult forms of glycine receptors with developmental changes in inhibitory synaptic receptor channels

Functional maturation of the nicotinic acetylcholine receptor is executed by its gamma-to-epsilon subunit switching. The glycine receptor also has fetal (alpha 2) and adult (alpha 1) isoforms. However, whether subunit switching is responsible for developmental changes in glycine receptor function is not known. We recorded single-channel currents from homomeric glycine receptors expressed in Xenopus oocytes with cRNAs encoding the alpha 2 or alpha 1 subunits and compared them with those recorded from native glycine receptors in rat spinal neurons at various ontogenic periods. The mean channel life times of the alpha 1 and mature glycine receptors were equally short, whereas both the alpha 2 and fetal receptors showed a significantly longer open time. Consistent with these results, the decay time of the glycinergic inhibitory postsynaptic currents (IPSCs) in spinal neurons became shorter during postnatal development. We conclude that developmental switching of alpha subunits may accelerate the kinetics of IPSCs.

vesl, a gene encoding VASP/Ena family related protein, is upregulated during seizure, long-term potentiation and synaptogenesis.

We have isolated a novel cDNA, vesl, that was induced during convulsive seizure in the rat hippocampus. The vesl gene encodes a protein of 186 amino acids that has significant homology to the EVH1 domain of the VASP/Ena family of proteins implicated in the control of microfilament dynamics. The expression of vesl mRNA was induced in the granule cell layer during persistent long‐term potentiation (LTP) of the dentate gyrus in an NMDA receptor‐dependent manner. Furthermore, vesl mRNA was expressed at a high level during hippocampal synaptogenesis. We suggest that the Vesl protein may be involved in the structural changes that occur at synapses during long‐lasting neuronal plasticity and development.

Cloning of a glycine receptor subtype expressed in rat brain and spinal cord during a specific period of neuronal development

Complementary (c) DNAs encoding a glycine receptor (GlyR) isomer were cloned from libraries constructed in λZAPII with poly (A)+ RNA of neonatal rat spinal cord. Northern blot analysis revealed that RNA hybridized to the cloned cDNA is detectable only for a period of late embryonic/ early postnatal stage of the spinal cord. Moreover, other central nervous tissues, such as hippocampus and cerebral cortex, in the infant rats are also rich in this message. The ‘neonatal (N) GlyR’ has 71% homology to that of another GlyR isoform in which adult rad cord is rich (AGlyR) Injection of a single RNA transcribed from the NGlyr‐cDNA into Xenopus oocyte induced functional formation of glycine‐gated Cl− channels, however, its pharmacological property differed from that of AGlyR.

Genome organization of the linear plasmid, pSKL, isolated from Saccharomyces kluyveri

SummaryWe have determined the complete nucleotide sequence of the linear DNA plasmid, pSKL, isolated from Saccharomyces kluyveri. Sequence analysis showed that pSKL has a high (A+T) content of 71.7%, and that there are 10 open reading frames (ORFs) larger than 250 nucleotides. All 10 ORFs were shown to be transcribed in S. kluyveri cells by S1 nuclease mapping analysis. The localization of ORFs, direction of transcription, and the predicted amino acid sequences of each ORF were quite similar to that of pGKL2, one of the killer plasmids found in Kluyveromyces lactis. The amino acid sequences of the largest two ORFs (ORF2 and ORF6) have homology with several DNA polymerases and RNA polymerases, respectively.

Down-regulation of glycine receptor channels by protein kinase C in Xenopus oocytes injected with synthetic RNA

Interaction of protein kinase C (PKC) with glycine receptor channels was examined using Xenopus oocytes expressing homomeric alpha 1 glycine channels. 4 beta-Phorbol 12-myristate 13-acetate (4 beta-PMA), an activator of PKC, reduced the response to glycine; this effect was inhibited in the presence of staurosporine, a PKC inhibitor. By contrast, 4 alpha-PMA, a poor PKC stimulant, did not affect the glycine currents. Thus, the PKC system is involved in negative-regulation of the glycine receptor channels. The results obtained from experiments with mutant receptors suggest that phosphorylation of the intracellular serine residue at 419 may relate to modification of the channel function.

Hairpin plasmid ― a novel linear DNA of perfect hairpin structure

The terminal structures of deletion derivatives of linear DNA killer plasmid from yeast were analyzed. The yeast Kluyveromyces lactis harbors two unique double‐stranded linear DNA killer plasmids, pGKL1 of 8.9 kb and pGKL2 of 13.4 kb. The killer toxin and the resistance to the killer are coded by pGKL1, while pGKL2 is required for the maintenance of pGKL1 in the cell. When the pGKL plasmids from K. lactis were transferred into Saccharomyces cerevisiae by transformation, non‐killer transformants harboring pGKL2 and new plasmids, F1 of 7.8 kb and F2 of 3.9 kb, were obtained. F2 was shown to be a linear DNA arising from a 5‐kb deletion of the right part of pGKL1. F1 was an inverted dimer of F2. Here we show that F2 has two different terminal structures: one end has a protein attached at the 5′ terminus whereas the two strands of duplex are linked together at the other end, thus forming a hairpin structure. This is a novel type of autonomously replicating DNA molecule.

Biologically active human and mouse nerve growth factors secreted by the yeast Saccharomyces cerevisiae

Nerve growth factor (NGF) is a trophic agent that is essential for the development and survival of sympathetic and sensory nerves. A chemically-synthesized DNA fragment encoding human NGF (hNGF) and a cDNA encoding mouse NGF (mNGF) were engineered for expression in the yeast, Saccharomyces cerevisiae. Expression and secretion of hNGF and mNGF was attempted under the direction of the yeast PGK promoter and with various leader sequences. Among the leader sequences tested, that of the yeast α-factor successfully directed secretion of both hNGF and mNGF that were correctly processed. The content of the recombinant NGF (reNGF) in the culture supernatant was estimated to be 1 μg/ml. The yeast-produced reNGF was able to bind to NGF receptors in rat pheochromocytoma (PC12) cells as efficiently as the standard mNGF, and partially purified reNGF could induce neurite outgrowth of PC12 cells. Thus, we have demonstrated that biologically active human and mouse reNGF can be produced in yeast cells.

Crystallization and preliminary X-ray crystallographic analysis of the receptor-uncoupled mutant of Gαi1

In order to understand the molecular mechanisms by which G-protein-coupled receptors (GPCRs) activate G proteins, the K349P mutant of Gαi1 (K349P), which is unable to couple to the muscarinic acetylcholine receptor, was prepared and its crystals were grown along with those of wild-type Gαi1 protein (WT). The two proteins were crystallized under almost identical conditions, thus enabling a detailed structural comparison. The crystallization conditions performed well irrespective of the identity of the bound nucleotide (GDP or GTPγS) and the crystals diffracted to resolutions of 2.2 A (WT·GDP), 2.8 A (WT·GTPγS), 2.6 A (K349P·GDP) and 3.2 A (K349P·GTPγS).

An efficient synthesis of polyguanylic acid by a thermophilic polynucleotide phosphorylase

Polyguanylic acid (poly(G)) was synthesized from GDP in a yield of 60-75% by Thermus thermophilus polynucleotide phosphorylase (polyribonucleotide: orthophosphate nucleotidyltransferase, EC 2.7.7.8) at 70 degrees C, pH 8.5 in the presence of Mg2+. The yield was dependent on the ratio of GDP to Mg2+, but was independent of the concentrations of enzyme or substrate. The maximal rate of GDP polymerization was obtained when the ratio of GDP to Mg2+ was 3:1. However, by prolonged incubation, the higher initial ratio of over 4:1 was preferred because of the rapid consumption of GDP in the reaction mixture. Poly(G) prepared by 1 h incubation was heterogeneous in size from 5 S to over 23 S, but by prolonged incubation of 19 h the size of product converged to 9 S as judged by sucrose density gradient centrifugation. Its chain length was determined by terminal nucleoside analysis to be 200 nucleotides long.

1417 A set of neural plasticity-regulated genes revealed by mRNA differential display

Neuropsin is a serine protease which relates several neuronal functrons such as development, synaptic organization and neurotransmission mechanisms. In the present study, we used in situ hybridization histochemistry and immunohistochemistry to analyze the precise localization of the hippocampal neuropsin. In situ hybridization, and immunohistochemical approaches showed that the neuropsin and its mRNA were localized in pyramidal cells of CA 1 -CA2 area of the ammons horn,but not in granular cells in the dentate gyrus. In kindling mice, the neuropsin mRNA and clearly increased in the pyramidal cells of ammon‘s horn. The activitydependent change of the neuropsin protein were also analyze with using Western blotting immunoprecipitation techniques.