Eric Kawashima

Tissue distribution of the P2X7 receptor.

The P2X7 receptor is a bifunctional molecule. The binding of ATP induces within milliseconds the opening of a channel selective for small cations, and within seconds a larger pore opens which allows permeation by molecules as large as propidium dyes (629 Da). In situ hybridization using a digoxigenin-labelled riboprobe, and immunohistochemistry using an antibody raised against a C-terminal peptide sequence, were used to determine the distribution of the P2X7 receptor mRNA and protein in rat and mouse tissues and cell lines. The brain of newborn rats showed a 6 kb RNA by Northern blotting, but this was not detectable in adult brain. By in situ hybridization and immunohistochemistry, there was heavy labelling of ependymal cells in both newborn and adult brain, but the brain parenchyma showed no labelling. However, P2X7 receptor-immunoreactive cells appeared in the penumbral region around an area of necrosis evoked by prior occlusion of the middle cerebral artery, suggesting expression of the receptor by activated microglia. NTW8 cells, a mouse microglial cell line, strongly expressed the P2X7 receptor mRNA and protein. The P2X7 receptor mRNA and protein were also observed in the majority of bone marrow cells, including those separately identified by their expression of other antigens as granulocytes, monocyte/macrophages and B lymphocytes. The expression of P2X7 receptor by brain macrophages rather than neurons would be consistent with a role in brain repair following inflammation, infarction or immune insult.

Organization of the mouse 5-HT3 receptor gene and functional expression of two splice variants

The structure of the mouse 5-HT3 receptor gene, 5-HT3R-A, is most similar to nicotinic acetylcholine receptor (nAChR) genes, in particular to the gene encoding the neuronal nAChR subunit alpha 7. These genes share among other things the location of three adjacent introns, suggesting that 5-HT3R-A and nAChR genes arose from a common precursor gene. The alternative use of two adjacent splice acceptor sites in intron 8 creates, in addition to the original 5-HT3R-A cDNA (5-HT3R-AL), a shorter isoform (5-HT3R-AS) which lacks six codons in the segment that translates into the major intracellular domain. This splice consensus sequence is not found in human genomic DNA. In mouse, we demonstrate by RNAse protection assay that 5-HT3R-AS mRNA is approximately 5 times more abundant than 5-HT3R-AL mRNA in both neuroblastoma cell lines and neuronal tissues. We used the Semliki Forest virus expression system for electrophysiological characterization of 5-HT3R-AS and 5-HT3R-AL in mammalian cells. No differences in electrophysiological characteristics, such as voltage dependence, desensitization kinetics, or unitary conductance were found between homomeric 5-HT3R-AS and 5-HT3R-AL receptors. Their properties are very similar to those of 5-HT3 receptors in mouse neuroblastoma cell lines.

DNA containing the base analogue 2-aminoadenine: preparation, use as hybridization probes and cleavage by restriction endonucleases

The base analogue 2-aminoadenine (2,6-diaminopurine, D) has been introduced at selected positions into synthetic oligodeoxyribonucleotides and DNA by the combined use of chemical and enzymatic methods. 2-aminoadenine substitution for adenine introduces changes in the minor groove of DNA and creates an additional hydrogen bond in the Watson-Crick base pair with thymine. Oligonucleotide hybridization probes containing 2-aminoadenine showed increased selectivity and hybridization strength during DNA-DNA hybridization to phage or genomic target DNA. Properties of the base analogue with respect to DNA modifying enzymes were examined. 2-aminoadenine was used to probe minor groove determinants during the treatment of DNA by 12 restriction endonucleases. Inhibition of cleavage was found for several restriction enzymes.

Expression and characterization of human D4 dopamine receptors in baculovirus-infected insect cells

The human D4 dopamine receptor has been genetically engineered for expression in insect cells using the baculovirus system. A D4 cDNA gene fusion construct [(1991) Nature 350, 610‐614] was synthetically modified to remove two introns from the coding region, and expressed in S. frugiperda (Sf9) cells as a fusion with a short sequence from the polyhedrin protein. Binding assays with [3H]spiperone indicated high levels of D4 receptor binding 90 h after infection and a pharmacological profile identical to that reported for D4 receptors expressed in COS‐7 cells using the cDNA gene hybrid. We also show that the agonist binding affinity of D4 receptors expressed in Sf9 cells can be shifted by GTP‐γ‐S, indicating coupling to G‐proteins.

Calcium influx and protein kinase Cα activation mediate arachidonic acid mobilization by the human NK‐2 receptor expressed in Chinese Hamster ovary cells

We have cloned a cDNA encoding the human ileal neurokinin‐2 (NK‐2) receptor which mediates powerful neurokinin A‐stimulated arachidonic acid (AA) and prostaglandin release when expressed in CHO cells. Two major signal transduction events appear to underlie this response. Firstly, AA liberation is critically dependent upon agonist‐stimulated influx of extracellular Ca2+ although not release from intracellular stores. Secondly, NK‐2 receptor‐linked AA mobilization requires concomitant PKC activation and based upon limited subtype immunodetectability as well as NKA‐stimulated translocation, PKCα could play a major role. While NKA‐stimulated Ca2+ mobilization is insensitive to preincubation with pertussis toxin, identical pretreatment inhibits AA release partially and blocks PKCa translocation completely. These observations indicate that in this cell system AA liberation reflects NK‐2 receptor‐dependent activation of two distinct but converging signal transduction pathways regulated by different G‐protein species and involving Ca2+ influx and PKCa activation.

Secondary structure at the bacteriophage G4 origin of complementary-strand DNA synthesis: in vivo requirements

The bacteriophage G4 origin of complementary strand DNA synthesis, G4 ori, contains several regions of potential secondary structure. In this study, we ask whether DNA secondary structure is important for G4 ori function in vivo. Point mutations were generated within a region of potential secondary structure so as to disrupt intrastrand base pairing. These mutations led to a strong temperature-dependent reduction in ori function in vivo. A double point mutation which introduces the same base substitutions without destabilizing intrastrand base pairing did not cause a temperature-dependent disruption in ori function. The double mutant did display a slight temperature-independent reduction in ori function compared to the wild-type G4 ori. Based on these findings, we conclude that DNA secondary structure, as well as recognition of specific sequences, is required for G4 ori activity in vivo.

Cloning and nucleotide sequence of the Salmonella typhimurium pepM gene

SummaryThe pepM gene coding for a methionine-specific aminopeptidase was cloned from Salmonella typhimurium and its nucleotide sequence determined. The gene encoded a 264 amino acid protein that was homologous to a similar protein from Escherichia coli. The sequence of an overproducer mutant allele, pepM100, contained a single base change in the likely −35 region of the pepM promoter that increased its homology to the consensus promoter sequence. A region downstream from the pepM coding sequence contained extensive inverted repeats and was homologous to sequences found elsewhere in both Salmonella and other bacterial species.