Antisense agrin cDNA transfection blocks neuroblastoma cell-induced acetylcholine receptor aggregation when co-cultured with myotubes

A neuroblastoma x glioma hybrid cell line, NG108-15, was able to induce the aggregation of AChRs when co-cultured with myotubes. NG108-15 cells in culture expressed agrin, producing a protein of similar to 220 kDa and a transcript of similar to 8.0 kb. The mRNA encoding the agrin isoform having no amino acid insertion at either the Y or the Z site, namely agrin(0,0), was the only transcript detected in NG108-15 cells when they were cultured alone or co-cultured with myotubes. NG108-15 cells could be induced to differentiate by chemical treatment, and the chemical-induced differentiation of NG108-15 cells increased the level of agrin mRNA expression approximately fourfold while the expression of a housekeeping gene remained relatively unchanged. The increase in agrin expression of differentiated NG108-15 cells paralleled the increase in AChR-aggregating activity of differentiated NG108-15 cells, indicating that the agrin derived from NG108-15 cells could be the receptor-aggregating factor. In addition, we created a stable clonal NG108-15 cell line that was transfected with antisense agrin cDNA and its expression of agrin was abolished, while its AChR-aggregating activity was completely lost when co-cultured with myotubes. This is the first direct demonstration that NG108-15 cell-induced AChR aggregation on cultured myotubes is mediated by neuron-derived agrin

Molecular diversity of 5S-rRNA spacer domain in Fritillaria species revealed by PCR analysis

Beimu (bulbs of Fritillaria) is an important traditional Chinese herbal medicine commonly used as an antitussive and expectorant. There are about 25 species and varieties of Fritillaria that carry the name Beimu on commercial markets. The price for each Beimu may differ by more than 100-fold. However, the identification of the origin of a particular species on the market is difficult. Here, a molecular method was used to identify various species of Fritillaria regardless of their geographical origin. The 5S-rRNA coding sequence is highly conserved in higher eukaryotes, but the spacer sequence of the 55-rRNA gene is variable among different species. Total genomic DNA from fresh leaves and bulbs of Fritillaria cirrhoso, F. puqiensis, F. anhuiensis, and F. thunbergii was extracted. The 5S-rRNA spacer region of the extracted DNAs was amplified by PCR with a pair of primers located within the conserved coding region. The isolated cDNA clones (similar to 600bp) covering the 5S-rRNA spacer domain were sequenced. By aligning the isolated nucleotide sequences of the four Fritillaria species, sequence diversity was found in the spacer region. Furthermore, a unique EcoR I site was used for the rapid identification of different species of Fritillaria. To our knowledge, this is the first report on the detection of 5S-rRNA spacer domain sequences of Fritillaria and their use to identify species

NG108-15 cells express neuregulin that induces AChR alpha-subunit synthesis in cultured myotubes

A cholinergic neuroblastoma x glioma hybrid cell line NG108-15 is able to form functional synapses, and contains both AChR-aggregating and AChR-inducing activities when cocultured with myotubes, Several lines of evidence indicate that the AChR-inducing activity of NG108-15 cells is derived from neuregulin, The conditioned medium of cultured NG108-15 cells induced the expression of AChR alpha-subunit as well as the tyrosine phosphorylation of erbB-3 receptor. NG108-15 cells expressed neuregulin with a protein of similar to 100 kDa in size and transcripts of similar to 6.8 kbp, similar to 2.6 kbp and similar to 1.8 kbp; mRNAs encoding beta 1 and alpha 2 isoforms of neuregulin were revealed. NG108-15 cells were induced to differentiate by chemicals, and the chemical-induced differentiation of NG108-15 cells increased the level of neuregulin mRNA expression similar to 3-fold while the expression of a housekeeping gene remained relatively unchanged, The activity of neuregulin in the conditioned medium of NG108-15 cells was reduced by treating the medium with heparin and anti-neuregulin antibody, In addition, NG108-15 tells were transfected with antisense neuregulin cDNA and its expression of neuregulin was reduced, while its neuregulin-induced tyrosine phosphorylation activity was markedly decreased. This is the first direct demonstration that the NG108-15 cell-induced AChR upregulation on cultured myotubes is mediated by neuron-derived neuregulin. (C) 1997 Federation of European Biochemical Societies

Expression and localization of endothelin converting enzyme in rat vas deferens

Endothelins (ETs) are a family of vasoconstrictor and mitogenic peptides originally isolated from the endothelial cells. Three isoforms of ET, namely ET-1, ET-2 and ET-3, are generated from their respective intermediate precursors big ETs through specific endoproteolytic cleavage by endothelin converting enzyme (ECE). Using reverse-transcription polymerase chain reaction (RT-PCR), we have isolated a cDNA encoding for ECE from both the prostatic and epididymal halves of rat vas deferens. In situ hybridization using digoxigenin-labeled ECE cDNA probe demonstrated that ECE mRNA is preferentially localized in the inner longitudinal smooth muscle layer adjacent to submucosa region of rat vas deferens. Both ET-1 and big ET-1 at 30 nM potentiated electrically stimulated contractile response of prostatic vas deferens. Pre-incubation of tissue with a metalloprotease ECE inhibitor phosphoramidon (10 mu M) strongly inhibited the response to big ET-1, but not to ET-1. On the other hand, big ET-1 failed to elicit contractile response of epididymal vas deferens. Phosphoramidon alone did not affect both the basal and electrically stimulated contractile responses in vas deferens. These data indicate that the circulating ET-1 and its immediate precursor big ET-1 could differentially regulate smooth muscle contractions in the prostatic and epididymal vas deferens of the rat

Exchange protein directly activated by cAMP 2 (Epac2)-deficient mice with anxiety and depression show defect in serotonergic system and neurogenesis

Poster Presentation 5 - B34: Neurotransmitters and Signalling Molecules - Monoamines - FENS-2148: no. B01

Species identification of Radix Astragali (Huangqi) by DNA sequence of its 5S-rRNA spacer domain

About 300 species and varieties of Astragalus are identified in China, making the identification of the origin of a particular Astragalus species on the consumer market difficult. A molecular genetic approach was developed to identify various species of Astragalus. Although the 5S-rRNA coding sequence is conserved in higher eukaryotes, the spacer domain of the 5S-rRNA gene has great diversity among different species. The 5S-rRNA spacer domain was amplified by polymerase chain reaction (PCR) from the isolated genomic DNA, and the PCR products (similar to 300 bp) covering the 5S-rRNA spacer domain were sequenced. The nucleotide sequences of Astragalus membranaceus, A. membranaceus var. mongholicus, A. lehmannianus, A. hoantchy, and of one closely related species Hedysarum polybotrys (Hongqi), were determined. Diversity in DNA sequence and restriction enzyme mapping among various species was found in their 5S-rRNA spacer domains. This is the first report on the detection of 5S-rRNA spacer region sequence of Astragalus, and the results could be used for genetic identification of Huangqi. (C) 2000 Elsevier Science Ltd. All rights reserved

A role of midkine in the development of the neuromuscular junction

Midkine (MK) is a member of a family of developmentally regulated neurotrophic and heparin-binding growth factors. It is expressed during the midgestation period in a retinoid-acid dependent manner during embryogenesis in the mouse, in vitro, it promotes neurite outgrowth from spinal cord neurons and cell migration. Ifs expression is strongest in the central nervous system, thus suggesting a function for this protein in neural development. in this study, the role of MK in synaptogenesis was examined in the Xenopus system. A Xenopus MK cDNA was cloned from an embryonic library encompassing neurulation and synaptogenesis stages. By Northern blot analysis, MK mRNA was detected from the onset of neurulation and throughout the stages of synaptogenesis in the Xenopus embryo. This suggests that MK is also an important growth regulator in Xenopus embryogenesis. To study the function of MK in the development of the neuromuscular junction (NMJ), fusion proteins were made and their ability to induce the formation of acetylcholine receptor (AChR) clusters in cultured muscle cells was studied. Beads coated with MK strongly induce AChR clustering. When nerve-muscle cocultures were labeled with antibodies made against the MK fusion protein, MK immunoreactivity was detected at the NMJ. Unlike heparin-binding growth-associated molecule (HB-GAM), another member of this growth factor family, MK expression cannot be detected in the muscle but is present in spinal cord neurites. Consistent with these in vitro data is the observation that MK mRNA is only localized in the central nervous system but the protein is deposited at the intersomitic junction where the NMJ is located in vivo. Exogenously applied MK does bind to the heparan sulfate proteoglycan on the surface of Xenopus muscle cells. Agrin, a heparan-sulfate proteoglycan that induces the formation of AChR clusters in cultured muscle cells, binds strongly to MK. Bath application of MK in conjunction with agrin results in a change in the pattern of AChR clustering induced by agrin alone. These data suggest that MK is a neuron-derived factor that participates in the signal transduction process during NMJ development