Genome-wide association identifies the T gene as a novel asthma pharmacogenetic locus.

RATIONALE: To date, most studies aimed at discovering genetic factors influencing treatment response in asthma have focused on biologic candidate genes. Genome-wide association studies (GWAS) can rapidly identify novel pharmacogenetic loci. OBJECTIVES: To investigate if GWAS can identify novel pharmacogenetic loci in asthma. METHODS: Using phenotypic and GWAS genotype data available through the NHLBI-funded Single-nucleotide polymorphism Health association-Asthma Resource Project, we analyzed differences in FEV(1) in response to inhaled corticosteroids in 418 white subjects with asthma. Of the 444,088 single nucleotide polymorphisms (SNPs) analyzed, the lowest 50 SNPs by P value were genotyped in an independent clinical trial population of 407 subjects with asthma. MEASUREMENTS AND MAIN RESULTS: The lowest P value for the GWAS analysis was 2.09 × 10(-6). Of the 47 SNPs successfully genotyped in the replication population, three were associated under the same genetic model in the same direction, including two of the top four SNPs ranked by P value. Combined P values for these SNPs were 1.06 × 10(-5) for rs3127412 and 6.13 × 10(-6) for rs6456042. Although these two were not located within a gene, they were tightly correlated with three variants mapping to potentially functional regions within the T gene. After genotyping, each T gene variant was also associated with lung function response to inhaled corticosteroids in each of the trials associated with rs3127412 and rs6456042 in the initial GWAS analysis. On average, there was a twofold to threefold difference in FEV(1) response for those subjects homozygous for the wild-type versus mutant alleles for each T gene SNP. CONCLUSIONS: Genome-wide association has identified the T gene as a novel pharmacogenetic locus for inhaled corticosteroid response in asthma.

Galantamine activates muscle-type nicotinic acetylcholine receptors without binding to the acetylcholine-binding site.

Galantamine (Reminyl; Janssen Pharmaceutica, Titusville, NJ) belongs to a class of acetylcholinesterase inhibitors approved for symptomatic treatment of Alzheimer's disease. The drug presumably acts by raising and prolonging the profile of acetylcholine (ACh) via an inhibitory effect on the esterase. However, there is also evidence demonstrating that galantamine can activate the nicotinic ACh receptor or modulate its activation by ACh. In this study, we have examined the ability of galantamine to directly activate the muscle-type nicotinic ACh receptor or to modulate receptor activation by selected nicotinic agonists. Studies of direct activation by galantamine demonstrated that this ligand is a low-efficacy agonist of the muscle-type ACh receptor. Point mutations in the M2-M3 linker (alphaS269I) and the M2 transmembrane domain (epsilonT264P) had similar effects on receptor activation by galantamine and nicotinic agonists, suggesting that the general features of receptor activation by galantamine are similar to that in the presence of ACh. Experiments performed in the simultaneous presence of galantamine and various nicotinic ligands showed that channel activation by the nicotinic ligands studied (ACh, carbachol, and choline) was not affected by the presence of galantamine at concentrations up to 100 microm. In addition, galantamine did not reduce the initial rate of binding for 125I-alpha-bungarotoxin. These results demonstrate that galantamine does not interfere with the occupation of the nicotinic agonist binding site by ACh, carbachol, or choline. We conclude that galantamine activates the muscle-type ACh receptor by interacting with a binding site that is distinct from the site for nicotinic agonists.

EGF regulates early embryonic mouse gut development in chemically defined organ culture.

The profound intestinal epithelial defects in the newborn epidermal growth factor receptor (EGFR) knockout mouse suggests that EGFR signaling plays important roles in embryonic gut development. Herein, we further elucidated the function of EGFR signaling on early embryonic gut development by comparing the effects of 1-10 ng/mL of exogenous epidermal growth factor (EGF) or 10-25 microM of the tyrphostin 3,4,5 trihydroxybenzene malononitrile, a specific inhibitor of EGFR tyrosine kinase, on intact E12 Swiss-Webster mouse midgut grown in chemically defined organ culture using Fitton-Jackson BGJb medium for 4 or 6 d. Intestinal development during culture was assayed by morphometry, histology, reverse transcription/competitive PCR for villin and intestinal fatty acid binding protein mRNA, and immunohistochemistry for epithelial proliferative markers. During organ culture, control specimens grew in length, developed smooth muscle, simple columnar epithelial and goblet cell phenotypes, showed early villus formation in the proximal intestine, and increased expression of villin and intestinal fatty acid binding protein mRNA. EGF failed to significantly alter small intestinal lengthening, whereas EGF 10 ng/mL inhibited colonic length growth. Tyrphostin 25 microM resulted in regional losses of stromal and smooth muscle cells in the small intestine and absent colonic goblet cells. In controls, cellular proliferation initially occurred throughout the small intestinal epithelium but became increasingly localized to the intervillus crypt regions. This sequestration of epithelial proliferation into crypts was much more apparent in EGF-treated versus tyrphostin-treated specimens. EGFR activation, therefore, appears to accelerate the maturation rate of goblet cells and the differential crypt/villus proliferation pattern in early embryonic mouse gut.

Basic fibroblast growth factor induces notochord formation and the expression of As-T, a Brachyury homolog, during ascidian embryogenesis.

The tadpole larva of an ascidian develops 40 notochord cells in the center of its tail. Most of the notochord cells originate from the A-line precursors, among which inductive interactions are required for the subsequent differentiation of notochord. The presumptive-endoderm blastomeres or presumptive-notochord blastomeres themselves are inducers of notochord formation. Notochord induction takes place during the 32-cell stage. In amphibia, mesoderm induction is thought to be mediated by several growth factors, for example, activins and basic fibroblast growth factor (bFGF). In the ascidian, Halocynthia roretzi, treatment with bFGF of presumptive-notochord blastomeres that had been isolated at the early 32-cell stage promoted the formation of notochord at a low concentration of bFGF (0.02 ng/ml), while activin failed to induce notochord differentiation. The effect of bFGF reached a maximum at the end of the 32-cell stage and rapidly faded at the beginning of the subsequent cleavage, the time for full induction of notochord being at least 20 minutes. The expression of As-T, a previously isolated ascidian homolog of the mouse Brachyury (T) gene, starts at the 64-cell stage and is detectable exclusively in the presumptive-notochord blastomeres. The present study showed that presumptive-notochord blastomeres, isolated at the early 32-cell stage, neither differentiated into notochord nor expressed the As-T gene. However, when the presumptive-notochord blastomeres were coisolated or recombined with inducer blastomeres, transcripts of As-T were detected. When presumptive-notochord blastomeres were treated with bFGF, the expression of the As-T gene was also detected. These results suggest that inductive interaction is required for the expression of the As-T gene and that the expression of the As-T gene is closely correlated with the determined state of the notochord-precursor cells.

Shift from fetal-type to Alzheimer-type phosphorylated Tau proteins in SKNSH-SY 5Y cells treated with okadaic acid.

Tau proteins are abnormally phosphorylated in Alzheimer's disease. Pathological Tau proteins named PHF-Tau 55, PHF-Tau 64, and PHF-Tau 69, are the main constituents of the paired helical filaments (PHF). When treating SKNSH-SY 5Y cells with okadaic acid (OA), Tau 55 protein was clearly induced whereas Tau 64 protein was only faintly induced. Here, we show that the absence of Tau 69 could be explained by the fact that adult isoforms containing N-terminal inserts are not detected. Phosphorylation is similar for untreated cellular Tau proteins and fetal Tau proteins, while OA cell treatment transformed fetal-type into Alzheimer-type phosphorylated proteins.