Exploratory aspirin resistance trial in healthy Japanese volunteers (J-ART) using platelet aggregation as a measure of thrombogenicity.

Aspirin prevents the production of thromboxane A2 (TXA2) by irreversibly inhibiting platelet cyclooxygenase, exhibiting antiplatelet actions. This agent has been reported to prevent relapse in patients with ischemic heart disease or cerebral infarction via this action mechanism. However, there are individual differences in this action, and aspirin is not effective in some patients, which is referred to as 'aspirin resistance'. In this study, we analyzed laboratory aspirin resistance by platelet aggregation in 110 healthy adult Japanese males using 24 single-nucleotide polymorphisms (SNPs) of nine genes involved in platelet aggregation/hemorrhage. Among SNPs involved in platelet aggregation, aspirin was less effective for 924T homozygote of a TXA2 receptor, 924T>C, and 1018C homozygote of a platelet membrane glycoprotein GPIbalpha, 1018C>T, suggesting that 924T and 1018C alleles are involved in aspirin resistance.

Substitution of Dmo1 with normal alleles results in decreased manifestation of diabetes in OLETF rats.

AIM: Dmo1 (Diabetes Mellitus OLETF type I) is a major quantitative trait locus for dyslipidaemia, obesity and diabetes phenotypes in the Otsuka Long Evans Tokushima Fatty (OLETF) rat strain. To evaluate possible metabolic and pathological improvements generated by correction of the Dmo1 genetic pathway, we produced congenic lines, in which both OLETF Dmo1 alleles are replaced by the F344-derived genome. METHODS: Congenic animals were produced by introgressing F344-derived Dmo1 alleles into the OLETF rat. Congenic animals of the fourth generation (BC4) were intercrossed to obtain F1 animals (BC4:F1). Animals of the next generation, BC4:F2, were used for this study. We used 23 BC4:F2 males harbouring homozygous replacement of the OLETF Dmo1 region with the F344-derived genome. Seven animals with OLETF-derived Dmo1 alleles were used as controls. RESULTS: Dmo1-F344/F344 congenic rats showed significant decreases in body weight, abdominal fat weight, serum triacylglycerols, total cholesterol, food consumption and blood glucose after glucose loading (13%, 39%, 45%, 27%, 18% and 27% respectively; p < 0.05) compared with Dmo1-OLETF/OLETF animals. Furthermore, histopathological analysis of the kidney showed that mesangial sclerosis, hyalin deposits and deposition of PAS-positive substance were significantly lower in Dmo1-F344/F344 animals (p < 0.05). CONCLUSION: Improvements in metabolic parameters and histopathological scores show that correction of the Dmo1 genetic pathway in the diabetic and mildly obese OLETF rat strain produces wide-ranging therapeutic effects. Thus, this pathway might represent a new drug target also applicable to humans.

Genetic analysis of pancreatic duct hyperplasia in Otsuka Long-Evans Tokushima Fatty rats: possible association with a region on rat chromosome 14 that includes the disrupted cholecystokinin-A receptor gene.

An Otsuka Long-Evans Tokushima Fatty (OLETF) strain of rat spontaneously developed hyperglycemia, hyperinsulinemia, insulin resistance and mild obesity, which had been studied as animal model for type II diabetes mellitus (T2DM). Recently, we observed that this strain coincidentally developed atypical hyperplasia of the choledocho-pancreatic ductal epithelium with a complete incidence. In an effort to locate genes responsible for this hyperplasia, we prepared 288 backcross progeny from a mating between OLETF rats and BN rats (which do not develop hyperplasia), and performed a genome-wide scan using 207 polymorphic genetic markers. We observed a prominent association of hyperplasia with a region involving a marker locus D14Mit4 (P = 0.00020, Fisher's exact test) and Cckar (the cholecystokinin-A receptor gene; P = 0.00025, Fisher's exact test) which is known to be disrupted in an OLETF strain. Our findings indicated that epithelial hyperplasia of the choledocho-pancreatic duct is associated with a region on rat chromosome 14 around the Cckar gene in an additive fashion with another two susceptible loci, each on chromosome 9 and 7. This implied the possibility that Cckar deficiency could result in a predisposition towards pancreatic duct hyperplasia.

Effects of Dmo1 on obesity, dyslipidaemia and hyperglycaemia in the Otsuka Long Evans Tokushima Fatty strain.

Whole-genome scans have identified Dmo1 as a major quantitative trait locus (QTL) for obesity and dyslipidaemia in the Otsuka Long Evans Tokushima Fatty (OLETF) rat. We have produced congenic rats for the Dmo1 locus, using marker-assisted speed congenic protocols, enforced by selective removal of other QTL regions (QTL-marker-assisted counterselection), to efficiently transfer chromosomal segments from non-diabetic Fischer 344 (F344) rats into the OLETF background. In the third generation of congenic animals, we observed a substantial therapeutic effect of the Dmo1 locus on lipid metabolism, obesity control and plasma glucose homeostasis. We conclude that single-allele correction of an impaired genetic pathway can generate a substantial therapeutic effect, despite the complex polygenic nature of type II diabetic syndromes.

Assessment of prediction accuracy of protein function from protein--protein interaction data.

Functional prediction of open reading frames coded in the genome is one of the most important tasks in yeast genomics. Among a number of large-scale experiments for assigning certain functional classes to proteins, experiments determining protein-protein interaction are especially important because interacting proteins usually have the same function. Thus, it seems possible to predict the function of a protein when the function of its interacting partner is known. However, in vitro experiments often suffer from artifacts and a protein can often have multiple binding partners with different functions. We developed an objective prediction method that can systematically include the information of indirect interaction. Our method can predict the subcellular localization, the cellular role and the biochemical function of yeast proteins with accuracies of 72.7%, 63.6% and 52.7%, respectively. The prediction accuracy rises for proteins with more than three binding partners and thus we present the open prediction results for 16 such proteins.

Conservation of the rat X chromosome gene order in rodent species.

We constructed the comparative cytogenetic maps of X chromosomes in three rodent species, Indian spiny mouse (Mus platythrix), Syrian hamster and Chinese hamster, using 26 mouse cDNA clones. Twenty-six, 22 and 22 out of the 26 genes, which were mapped to human, mouse and rat X chromosomes in our previous study, were newly localized to X chromosomes of Indian spiny mouse, and Syrian and Chinese hamsters, respectively. The order of the genes aligned on the long arm of human X chromosome was highly conserved in rat and the three rodent species except mouse. The present results suggest a possibility that the rat X chromosome retains the ancestral form of the rodent X chromosomes.

Automated extraction of information on protein-protein interactions from the biological literature.

MOTIVATION: To understand biological process, we must clarify how proteins interact with each other. However, since information about protein-protein interactions still exists primarily in the scientific literature, it is not accessible in a computer-readable format. Efficient processing of large amounts of interactions therefore needs an intelligent information extraction method. Our aim is to develop an efficient method for extracting information on protein-protein interaction from scientific literature. RESULTS: We present a method for extracting information on protein-protein interactions from the scientific literature. This method, which employs only a protein name dictionary, surface clues on word patterns and simple part-of-speech rules, achieved high recall and precision rates for yeast (recall = 86.8% and precision = 94.3%) and Escherichia coli (recall = 82.5% and precision = 93.5%). The result of extraction suggests that our method should be applicable to any species for which a protein name dictionary is constructed. AVAILABILITY: The program is available on request from the authors.

Single-allele correction of the Dmo1 locus in congenic animals substantially attenuates obesity, dyslipidaemia and diabetes phenotypes of the OLETF rat.

1. Whole-genome scans have identified Dmo1 as a major quantitative trait locus for dyslipidaemia and obesity in the Otsuka Long Evans Tokushima Fatty (OLETF) rat. 2. We have produced congenic rats for the Dmo1 locus through successive back-cross breeding with diabetic OLETF rats. Marker-assisted speed congenic protocols were applied to efficiently transfer chromosomal segments from non-diabetic Brown Norway (BN) rats into the OLETF background. 3. In the fourth generation of congenic animals, we observed a substantial therapeutic effect of the Dmo1 locus on lipid metabolism, obesity control and plasma glucose homeostasis. 4. We have concluded that Dmo1 primarily affects lipid homeostasis, obesity control and/or glucose homeostasis at fasting and is secondarily involved in glucose homeostasis after loading. 5. The results of the present study show that single-allele correction of a genetic defect of the Dmo1 locus can generate a substantial therapeutic effect, despite the complex polygenic nature of type II diabetic syndromes.

Quantitative trait loci for lipid metabolism in the study of OLETF x (OLETF x Fischer 344) backcross rats.

1. The Otsuka Long-Evans Tokushima Fatty (OLETF) rat is a model of type II diabetes with accompanying dyslipidaemia and obesity. 2. To define chromosomal intervals associated with obesity (abdominal fat weight and plasma leptin levels), dyslipidaemia (plasma triglyceride, cholesterol and free fatty acids) and hyperglycaemia (plasma glucose levels), we have performed genome-wide quantitative traits loci (QTL) analyses of 115 male OLETF x (OLETF x Fischer 344) backcross animals at 16 weeks of age. 3. The Diabetes Mellitus OLETF type I (Dmo1) locus on rat chromosome 1 showed statistically significant involvement in elevations of plasma levels of triglycerides (P = 4.87 x 10(-6) at D1Rat90) and total cholesterol (P = 1.16 x 10(-5) at D1Rat306). 4. No other loci produced significant linkage to these observed phenotypes. 5. These analyses have confirmed the importance of Dmo1 in lipid homeostasis at younger ages as well as during overt diabetes, which appears later. Thus, alterations at the Dmo1 locus are a major risk factor for pathogenesis in the strain, a finding that agrees with physiological studies that indicate a role for dyslipidaemia in the type II diabetic syndrome of OLETF rats.

Mining the quantitative trait loci associated with oral glucose tolerance in the OLETF rat.

Although the synergetic effects of multiple marker loci regarding quantitative traits such as blood glucose level have attracted interest, previous conclusions have been based on assumptions that each marker locus behaves independently of the other, leading to approximation. To cope with this problem, this paper focuses on the effects of multiple genetic factors and tries to find significant marker combinations by using conjunctive rules regarding genotypes at multiple marker loci. Application of the proposed method on the OLETF model rat of non-insulin dependent diabetes mellitus (NIDDM) has found significant combinations of marker loci with respect to oral glucose tolerance (OGT).

Characterization of newly developed SSLP markers for the rat.

We have isolated more than 12,000 clones containing microsatellite sequences, mainly consisting of (CA)n dinucleotide repeats, using genomic DNA from the BN strain of laboratory rat. Data trimming yielded 9636 non-redundant microsatellite sequences, and we designed oligonucleotide primer pairs to amplify 8189 of these. PCR amplification of genomic DNA from five different rat strains yielded clean amplification products for 7040 of these simple-sequence-length-polymorphism (SSLP) markers; 3019 markers had been mapped previously by radiation hybrid (RH) mapping methods (Nat Genet 22, 27-36, 1998). Here we report the characterization of these newly developed microsatellite markers as well as the release of previously unpublished microsatellite marker information. In addition, we have constructed a genome-wide linkage map of 515 markers, 204 of which are derived from our new collection, by genotyping 48 F2 progeny of (OLETFxBN)F2 crosses. This map spans 1830.9 cM, with an average spacing of 3.56 cM. Together with our ongoing project of preparing a whole-genome radiation hybrid map for the rat, this dense linkage map should provide a valuable resource for genetic studies in this model species.

Genetic dissection of "OLETF," a rat model for non-insulin-dependent diabetes mellitus: quantitative trait locus analysis of (OLETF x BN) x OLETF.

To identify genetic determinants relevant to non-insulin-dependent diabetes mellitus (NIDDM), we performed a genome-wide analysis for quantitative trait loci (QTLs) using 359 backcross progeny of the Otsuka Long-Evans Tokushima Fatty (OLETF) rat. The OLETF strain is a well-studied animal model of obese NIDDM, with features of hyperinsulinemia, hyperglycemia, insulin resistance, and abundant abdominal fat. Our extensive genomic scanning with 218 markers revealed nine significant QTLs, including a strong determinant of obesity on chromosome 1 (Dmo1: LOD = 13.99, for body weight). Two highly significant QTLs for glucose homeostasis were found, one on chromosome 1 (Dmo4 LOD = 7.16, for postprandial glucose level) and the other on chromosome X (Dmo11/Odb1: LOD = 7.81, for postprandial glucose level). These data are comparable to results of our previous studies of the OLETF rat.

A radiation hybrid map of the rat genome containing 5,255 markers.

A whole-genome radiation hybrid (RH) panel was used to construct a high-resolution map of the rat genome based on microsatellite and gene markers. These include 3,019 new microsatellite markers described here for the first time and 1,714 microsatellite markers with known genetic locations, allowing comparison and integration of maps from different sources. A robust RH framework map containing 1,030 positions ordered with odds of at least 1,000:1 has been defined as a tool for mapping these markers, and for future RH mapping in the rat. More than 500 genes which have been mapped in mouse and/or human were localized with respect to the rat RH framework, allowing the construction of detailed rat-mouse and rat-human comparative maps and illustrating the power of the RH approach for comparative mapping.

Tracing Synergetic Behavior of the QTLs Affecting Oral Glucose Tolerance in the OLETF Rat.

The synergetic effects of multiple marker loci regarding quantitative traits such as blood glucose level have attracted interest. In the OLETF model rat of non-insulin dependent diabetes mellitus (NIDDM), our previous study focusing on the effects of multiple genetic factors has found significant marker combinations with respect to oral glucose tolerance (OGT) at 60 minutes after oral administration. Besides the interaction among markers at a particular time point, their correlated behavior in a time series is another interest. Based on the previous results, in this paper, we report the behavior of markers in a time series by using a series of measurements of OGT.

Comparative FISH mapping of mouse and rat homologues of twenty-five human X-linked genes.

We constructed a comparative cytogenetic map of 25 functional genes in mouse and rat X chromosomes by direct R-banding fluorescence in situ hybridization. Nineteen and 22 out of the 25 genes, which have been mapped on the human X chromosome, were newly localized to mouse and rat X chromosomes, respectively. Twenty-two additional genes were integrated in the rat-mouse-human comparative map of the X chromosome in this study. Comparison of the gene order indicated the presence of four chromosome segments with conserved linkage homology between mouse and rat X chromosomes, suggesting that a minimum of four chromosomal inversion events occurred between mouse and rat X chromosomes during the evolution of the two species. Four chromosome segments with conserved linkage homology were found between human and rat X chromosomes.

Genetic dissection of "OLETF", a rat model for non-insulin-dependent diabetes mellitus.

To elucidate the genetic factors underlying non-insulin-dependent diabetes mellitus (NIDDM), we performed genome-wide quantitative trait locus (QTL) analysis, using the Otsuka Long-Evans Tokushima Fatty (OLETF) rat. The OLETF rat is an excellent animal model of NIDDM because the features of the disease closely resemble human NIDDM. Genetic dissection with two kinds of F2 intercross progeny, from matings between the OLETF rat and non-diabetic control rats F344 or BN, allowed us to identify on Chromosome (Chr) 1 a major QTL associated with features of NIDDM that was common to both crosses. We also mapped two additional significant loci, on Chrs 7 and 14, in the (OLETF x F344)F2 cross alone, and designated these three loci as Diabetes mellitus, OLETF type Dmo 1, Dmo2 and Dmo3 respectively. With regard to suggestive QTLs, we found loci on Chrs 10, 11, and 16 that were common to both crosses, as well as loci on Chrs 5 and 12 in the (OLETF x F344)F2 cross and on Chrs 4 and 13 in the (OLETF x BN)F2 cross. Our results showed that NIDDM in the OLETF rat is polygenic and demonstrated that different genetic backgrounds could affect "fitness" for QTLs and produce different phenotypic effects from the same locus.

Cloning and characterization of a novel member of the human Mad gene family (MADH6).

MAD (mothers against decapentaplegic)-related proteins (MADRs) are intracellular components that play critical roles in signal-transduction pathways involving the transforming growth factor beta (TGFbeta) superfamily. Some Mad genes are candidates for tumor-suppressor functions. From a human fetal brain cDNA library we have isolated a novel Mad-related gene. Two alternatively transcribed mRNAs encode deduced 430- and 467-amino-acid peptides that showed high levels of similarity to MADR1/Smad1/hMAD1 (about 80% identity at the amino acid level). This gene, which we designated MADH6, resides on 13q12-q14 between BRCA2 and RB, a region that frequently displays loss of heterozygosity in breast, liver, and prostate cancers.

Isolation and mapping of karyopherin alpha 3 (KPNA3), a human gene that is highly homologous to genes encoding Xenopus importin, yeast SRP1 and human RCH1.

From a human fetal-brain cDNA library, we isolated and characterized a novel gene (KPNA3) encoding a protein highly homologous to certain nuclear transport proteins of Xenopus and human. The complete cDNA clone, designated karyopherin alpha 3, contained an open reading frame of 1,563 nucleotides encoding 521 amino acids. The predicted amino acid sequence showed 48%, 45% and 48% identity with Xenopus importin, yeast SRP1 and human RCH1, respectively. The similarities among these proteins suggest that karyopherin alpha 3 may be involved in the nuclear transport system. Eight repeats of the arm motif were well conserved among these proteins. The N-terminal region of the predicted karyopherin alpha 3 product was highly basic and the C-terminal region was strongly acidic. A 4.3-kb transcript was expressed in all adult human tissues examined by Northern blotting. The cDNA clone was assigned to chromosome band 13q14.3 by fluorescence in situ hybridization.

Applying Association Rule Discovery Algorithm to Multipoint Linkage Analysis.

Knowledge discovery in large databases (KDD) is being performed in several application domains, for example, the analysis of sales data, and is expected to be applied to other domains. We propose a KDD approach to multipoint linkage analysis, which is a way of ordering loci on a chromosome. Strict multipoint linkage analysis based on maximum likelihood estimation is a computationally tough problem. So far various kinds of approximate methods have been implemented. Our method based on the discovery of association between genetic recombinations is so different from others that it is useful to recheck the result of them. In this paper, we describe how to apply the framework of association rule discovery to linkage analysis, and also discuss that filtering input data and interpretation of discovered rules after data mining are practically important as well as data mining process itself.