Complete sequence of a cDNA encoding an active rat choline acetyltransferase: A tool to investigate the plasticity of cholinergic phenotype expression

Abstract

A cDNA clone encoding the complete sequence of an active rat choline acetyltransferase (ChoAcTase; acetyl-CoA:choline O-acetyltransferase, EC 2.3.1.6) has been isolated. Analysis of the deduced amino acid sequence reveals 85% and 31% identity with the porcine and Drosophila melanogaster enzymes, respectively. To further elucidate the molecular basis of neurotransmitter-related phenotypic plasticity, the expression of ChoAcTase mRNA was compared with that of tyrosine hydroxylase [TH; tyrosine 3-monooxygenase, L-tyrosine, tetrahydropteridine:oxygen oxidoreductase (3-hydroxylating), EC 1.14.16.2], in neurons from superior cervical ganglia grown in the following conditions: 1) normal medium, 2) high K+ medium, and 3) normal medium supplemented with 50% muscle-conditioned medium (CM). TH mRNA was expressed in all three media; its level rose in high K+ and decreased strikingly in the presence of CM. ChoAcTase mRNA could be visualized in CM, but fell to undetectable levels in normal and high K+ media. These results suggest that translational or post-translational mechanisms do not play a major role for the modulation of neurotransmitter-associated phenotype.

Muscarinic (m2/m4) receptors reduce N- and P-type Ca2+ currents in rat neostriatal cholinergic interneurons through a fast, membrane- delimited, G-protein pathway

“…The ChAT mRNA (Brice et al, 1989) was identified using a pair of primers flanking a splicing site near the 3' terminus of the coding region. The upper primer was 5'-ATG GCC ATT GAC AAC CAT CTT CTG (nucleotides 1729-1752) and located on exon 14.…”

Beta-amyloid expression, release and extracellular deposition in aged rat brain slices

“…Western blot analysis of 100 ng rat b-amyloid(1-42) showed a strong single band of 4 kDa and a weaker band at 12 kDa under reducing or non-reducing conditions. Western blot analysis of 100 ng sbAPP protein showed a strong band of approximately 100 kDa under non-reducing conditions with the anti-APP A4 antibody, whereas no sbAPP-protein (1000 ng) was detectable with the anti-b-amyloid (17)(18)(19)(20)(21)(22)(23)(24) antibody. Brain slices incubated for 12 or 16 weeks at physiological pH tested conditions (pH 7.2 or pH 6.0, 7ApoE4, 2 weeks 371C) when the mix was loaded directly onto the gel.…”

“…18 Briefly, brain slices were transferred onto slides (ProbeOn slides, Fisher Biotech, Pittsburgh, PA, USA) and frozen in a CO 2 stream and stored at À201C until use. Antisense oligonucleotides complementary to nucleotide base pairs 1818-1860 of the choline acyteltransferase (ChAT) gene (43mer) 19 and the APP695 gene (40mer) 20 were labeled at the 3 0 end with [a- 35 S]dATP using terminal deoxyribonucleotidyl transferase (New England Nuclear, Vienna, Austria) and purified using Qiagen nucleotide removal kit (Qiagen, Vienna, Austria). Sections were hybridized at 421C overnight in a humidified chamber with 0.1 ml per slide of a hybridization solution (50% formamide, 4 Â SSC, 0.02% polyvinylpyrrolidone, 0.02% Ficoll, 0.02% bovine serum albumin, 10% dextran sulfate, 0.5 mg/ml sheared salmon sperm DNA, 1% sarcosyl (N-lauroyl sarcosine), 0.02 M NaPO4 (pH 7.0), 50 mM dithiothreitol) containing 1 Â 10 7 c.p.m./ml probe.…”

“…For the detection the Western Breeze Chromogenic System (Invitrogen) was used. Briefly, blots were blocked for 30 min with blocking buffer, then incubated for 90-180 min with the primary anti-b-amyloid (17)(18)(19)(20)(21)(22)(23)(24) antibody (Chemicon; 1:1000) or mouse anti-APP A4 (Chemicon MAB348; 1:2000), washed and incubated with alkaline phosphatase-conjugated anti-mouse IgG (Invitrogen) for 30-60 min at room temperature. After being washed, bound antibodies were visualized by p-nitro blue tetrazolium chloride and 5-bromo-4-chloro-3-indolyl phosphate (both Roche Molecular Biochemicals, Vienna, Austria).…”

“…Western blot of rat b-amyloid(1-42) and APP A4 Western Blot analysis for rat b-amyloid (17)(18)(19)(20)(21)(22)(23)(24) and sbAPP is shown in Figure 7. Western blot analysis of 100 ng rat b-amyloid(1-42) showed a strong single band of 4 kDa and a weaker band at 12 kDa under reducing or non-reducing conditions.…”