A horseshoe crab receptor structurally related to Drosophila Toll.

Innate immunity against microbial pathogens relies on the pattern recognition of cell wall components on invading microbes. Recent evidence has shown that a mammalian Toll-like receptor (TLR) is activated by bacterial lipopolysaccharides (LPS). The innate immunity in invertebrates is also triggered by LPS, as seen in the hemolymph coagulation in horseshoe crab. We report the cloning of a TLR from the Japanese horseshoe crab Tachypleus tridentatus. A cDNA coding for Tachypleus Toll was isolated from a hemocyte cDNA library and the open reading frame codes for a proprotein including a signal sequence. Like Drosophila Toll, Tachypleus Toll is a type I transmembrane protein with an extracellular domain consisting of two leucine-rich repeats flanked by two cystein-rich clusters and a cytoplasmic domain exhibiting striking similarity with the cytoplasmic domain of interleukin-1 receptor. Tachypleus Toll is most similar to Drosophila Toll in the domain architecture and the overall length.

[Studies on an antibody-coated tube method of aldosterone].

We studied on the basic performance of a non-extraction coated tube RIA kit, DPC Aldosterone kit (Nippon DPC Corporation). 1. The average binding CVs of the calibrators (25 to 1,200 pg/ml) were stable in a range from 1.4 to 1.9%. 2. The minimum sensitivity was 17 pg/ml from 2SD method of 0 calibrator. 3. The intra-assay reproducibility test showed an average CV as 9.0%, and the inter-assay test showed as 8.8%. 4. The recovery ratios of a recovery test were 85.4 to 121.6%. 5. The correlation test with another maker's kit showed a correlation curve as y = 0.970 x + 19.68 and a correlation coefficiency as r = 0.970. 6. The normal range, the correlation between fasting recumbency and daytime sitting, and the correlation between walking and resting, showed similar results as former reports.

[Evaluation of a radioimmunoassay of alpha 1-microglobulin (alpha 1-m) with simplified procedures].

A newly established double antibody radioimmunoassay (RIA) was fundamentally and clinically evaluated. Original procedures were partially modified as follows: Sample volume for serum and urine was changed to 25 microliters, and thus 200 mg/l of alpha 1-m standard was prepared using 50 microliters of original standard solution (100 mg/l). The results were satisfactory in sensitivity (0.3 mg/l obtained from -2SD method), intra-assay precision with its coefficient variation (CV) ranging from 3.0 to 7.4%, interassay precision with its CV ranging from 3.0 to 10.7%, and recovery with the mean value of 102.4% in serum and 108.2% in urine respectively. There were no changes about alpha 1-m value between diluted (2 times) and undiluted with high concentration samples. Normal levels of alpha 1-m were less than 25 mg/l in serum and less than 10 mg/l in urine. The present results indicate that the determination of alpha 1-m could be very simple and useful for the most sensitive screening test for the evaluation of renal function.

High-density single-nucleotide polymorphism (SNP) map of the 150-kb region corresponding to the human ATP-binding cassette transporter A1 (ABCA1) gene.

Highly dense catalogs of human genetic variations, in combination with high-throughput genotyping technologies, are expected to clarify individual genetic differences in pharmacological responsiveness and predispositions to common diseases. Here we report single-nucleotide polymorphisms (SNPs) present among 48 Japanese individuals at the locus for the human ATP-binding cassette transporter A1 (ABCA1) gene. ABCA1 plays a key role in apolipoprotein-mediated cholesterol transport, and mutations in this gene are responsible for Tangier disease and familial high-density lipoprotein deficiency associated with reduced cholesterol efflux. We identified a total of 162 SNPs, 149 of which were novel, within the 150-kb region encompassing the entire ABCA1 gene. Eight of the SNPs lie within coding elements, two in 5' flanking regions, 147 in introns, and five in 3' untranslated regions, but none were found in 5' untranslated or 3' flanking regions. The ratio of transitions to transversions was approximately 2.37 to 1. Our dense SNP map of this region could serve as a powerful resource for studies of complex genetic diseases that may be associated with ABCA1 and of individual responses to drug therapy.

Catalog of 46 single-nucleotide polymorphisms (SNPs) in the microsomal glutathione S-transferase 1 (MGST1) gene.

A major goal in our laboratory is to understand the role of common genetic variations among individual patients as regards susceptibility to common diseases and differences in therapeutic efficacy and/or side effects of drugs. As an addition to the high-density SNP (single-nucleotide polymorphism) maps of 12 glutathione S-transferase and related genes reported earlier, we provide here an SNP map of the microsomal glutathione S-transferase 1 (MGST1) gene. Among 48 healthy Japanese volunteers examined. we identified a total of 46 SNPs at this locus, 36 of which had not been reported before: 4 in the promoter region, 34 in introns, 3 in the 3' untranslated region, and 5 in the 3' flanking region. No SNP was found in 5'untranslated or coding regions. The ratio of transition to transversion was approximately 1.2:1. Among the 13 insertion-deletion polymorphisms was a 2-bp deletion in the coding region of MGST1 in DNA from one of the volunteers, which resulted in a frame-shift mutation. Since the gene product encoded by this mutant allele would lack the C-terminal half including the MAPEG (membrane-associated proteins in eicosanoid and glutathione metabolism) domain, MGST1 activity is likely to be reduced in the carrier's cells. The SNP map presented here adds to the archive of tools for studying complex genetic diseases, population migration patterns, and a variety of pharmacogenetic possibilities.

Catalog of 258 single-nucleotide polymorphisms (SNPs) in genes encoding three organic anion transporters, three organic anion-transporting polypeptides, and three NADH:ubiquinone oxidoreductase flavoproteins.

We present here a series of high-density maps of single-nucleotide polymorphisms (SNPs) detected in genes encoding three organic-anion transporters, three organic anion-transporting polypeptides, and three nicotinamide adenine dinucleotide, reduced:ubiquinone oxidoreductase flavoproteins. A total of 258 SNPs were identified among these nine genes through systematic screening of DNA from 48 Japanese individuals: 17 in 5' flanking regions, three in 5' untranslated regions, 13 in coding regions, 211 in introns, six in 3' untranslated regions, and 8 in 3' flanking regions. By comparing our data with SNPs deposited in the dbSNP database in the National Center for Biotechnology Information, we determined that 236 (91.5%) were novel. In addition, 46 genetic variations of other types were discovered within these loci. These high-resolution maps will serve as a useful resource for analyzing potential associations between variations in these nine genes and differences in human susceptibilities to common diseases or response to drug therapies.

Catalog of 320 single nucleotide polymorphisms (SNPs) in 20 quinone oxidoreductase and sulfotransferase genes.

Single nucleotide polymorphisms (SNPs) in genes encoding drug-metabolizing enzymes, transporters, receptors, and other drug targets have been widely implicated as contributors to differences among individuals as regards the efficacy and toxicity of many medications, as well as the susceptibility to complex diseases. By combining the polymerase chain reaction (PCR) technique with direct sequencing, we screened genomic DNAs from 48 Japanese volunteers for SNPs in genes encoding three quinone oxidoreductases (NQO1, NQO2, and PIG3) and 17 sulfotransferases (SULT1A1, SULT1A2, SULT1A3, SULT1C1, SULT1C2, SULT2A1, SULT2B1, ST1B2, TPST1, TPST2, SULTX3, STE, CST, HNK-1 ST, CHST2, CHST4, and CHST5). In all, we identified 320 SNPs from these 20 loci: 22 within coding elements, 21 in 5' flanking regions, 10 in 5' untranslated regions, 223 in introns, 19 in 3' untranslated regions, and 25 in 3' flanking regions. The ratio of transitions to transversions was approximately 2.3 to 1. Of the 22 coding SNPs, 6 were nonsynonymous substitutions that resulted in amino-acid substitutions. The high-density SNP maps we constructed from this data for each of the quinone oxidoreductases and sulfotransferases examined here should provide useful information for investigations designed to detect association(s) between genetic variations and common diseases or responsiveness to drug therapy.

Catalog of 434 single-nucleotide polymorphisms (SNPs) in genes of the alcohol dehydrogenase, glutathione S-transferase, and nicotinamide adenine dinucleotide, reduced (NADH) ubiquinone oxidoreductase families.

An approach based on development of a large archive of single-nucleotide polymorphisms (SNPs) throughout the human genome is expected to facilitate large-scale studies to identify genes associated with drug efficacy and side effects, or susceptibility to common diseases. We have already described collections of SNPs present among various genes encoding drug-metabolizing enzymes. Here we report SNPs for such enzymes at additional loci, including 8 alcohol dehydrogenases, 12 glutathione S-transferases, and 18 belonging to the NADH-ubiquinone oxidoreductase family. Among DNA samples from 48 Japanese volunteers, we identified a total of 434 SNPs at these 38 loci: 27 within coding elements, 52 in 5' flanking regions, five in 5' untranslated regions, 293 in introns, 20 in 3' untranslated regions, and 37 in 3' flanking regions. The ratio of transitions to transversions was approximately 2.1 to 1. Among the 27 coding SNPs, 13 were nonsynonymous changes that resulted in amino acid substitutions. Our collection of SNPs derived from this study should prove useful for investigations designed to detect associations between genetic variations and common diseases or responsiveness to drug therapy.