Variations in the C3, C3a receptor, and C5 genes affect susceptibility to bronchial asthma

Bronchial asthma (BA) is a common chronic inflammatory disease characterized by hyperresponsive airways, excess mucus production, eosinophil activation, and the production of IgE. The complement system plays an immunoregulatory role at the interface of innate and acquired immunities. Recent studies have provided evidence that C3, C3a receptor, and C5 are linked to airway hyperresponsiveness. To determine whether genetic variations in the genes of the complement system affect susceptibility to BA, we screened single nucleotide polymorphisms (SNPs) in C3, C5, the C3a receptor gene (C3AR1), and the C5a receptor gene (C5R1) and performed association studies in the Japanese population. The results of this SNP case-control study suggested an association between 4896C/T in the C3 gene and atopic childhood BA (P=0.0078) as well as adult BA (P=0.010). When patient data were stratified according to elevated total IgE levels, 4896C/T was more closely associated with adult BA (P=0.0016). A patient-only association study suggested that severity of childhood BA was associated with 1526G/A of the C3AR1 gene (P=0.0057). We identified a high-risk haplotype of the C3 gene for childhood (P=0.0021) and adult BA (P=0.0058) and a low-risk haplotype for adult BA (P=0.00011). We also identified a haplotype of the C5 gene that was protective against childhood BA (P=1.4×10^–6) and adult BA (P=0.00063). These results suggest that the C3 and C5 pathways of the complement system play important roles in the pathogenesis of BA and that polymorphisms of these genes affect susceptibility to BA.     

Update of mouse microsatellite database of Japan (MMDBJ).

We updated a database of microsatellite marker polymorphisms found in inbred strains of the mouse, most of which were derived from the wild stocks of four Mus musculus subspecies, M. m. domesticus, M. m. musculus, M. m.castaneus and M. m. molossinus. The major aim of constructing this database was to establish the genetic status of these inbred strains as resources for linkage analysis and positional cloning. The inbred strains incorporated in our database are A/J, C57BL/6J, CBA/J, DBA/2J, SM/J, SWR/J, 129Sv/J, MSM/Ms, JF1/Ms, CAST/Ei, NC/Nga, BLG2/Ms, NJL/Ms, PGN2/Ms, SK/CamEi and SWN/Ms, which have not or have only been poorly incorporated in the Whitehead Institute/MIT (WI/MIT) microsatellite database. The number of polymorphic microsatellite loci incorporated in our database is over 1,000 in all strains, and the URL site for our database is located at http:// www.shigen.nig.ac.jp /mouse/mmdbj/mouse.html.     

Protein kinase A determines timing of early differentiation through epigenetic regulation with G9a

Timing of cell differentiation is strictly controlled and is crucial for normal development and stem cell differentiation. However, underlying mechanisms regulating differentiation timing are fully unknown. Here, we show a molecular mechanism determining differentiation timing from mouse embryonic stem cells (ESCs). Activation of protein kinase A (PKA) modulates differentiation timing to accelerate the appearance of mesoderm and other germ layer cells, reciprocally correlated with the earlier disappearance of pluripotent markers after ESC differentiation.?PKA activation increases protein expression of G9a, an H3K9 methyltransferase, along with earlier H3K9 dimethylation and DNA methylation in Oct3/4 and Nanog gene promoters. Deletion of G9a completely abolishes PKA-elicited acceleration of differentiation and epigenetic modification. Furthermore, G9a knockout mice show prolonged expressions of?Oct3/4 and Nanog at embryonic day 7.5 and delayed development. In this study, we demonstrate molecular machinery that regulates timing of multilineage differentiation by linking signaling with epigenetics.     

Cloning and chromosomal mapping of the mouse DNA-dependent protein kinase gene

 Severe combined immune deficiency (scid) mice are assumed to have two types of abnormalities: one is high radiosensitivity and the other is abnormal recombination in immunoglobulin and T-cell receptor genes. The human chromosome 8 q1.1 region has an ability to complement the scid aberrations. Moreover, the localization of the subunit DNA-dependent protein kinase [DNA-PK_cs] participating in DNA double-strand break repair in the same locus was clarified. In scid mouse cells, the number of DNA-PK_cs products and extent of DNA-PK activity remarkably decrease. These observations gave rise to the assumption that DNA-PK_cs is the scid factor itself. In order to determine whether the DNA-PK _cs gene is the scid gene, we isolated the mouse DNA-PK _cs gene and investigated its chromosomal locus by fluorescence in situ hybridization (FISH). Consequently, it became clear that the mouse DNA-PK _cs gene existed in the centromeric region of mouse chromosome 16, determined by cross-genetic study, as a scid locus. This finding strongly suggests that mouse DNA-PK _cs is the scid gene. Received: 22 March 1996     

Dual loading of the fluorescent indicator fura-2 and 2,7-biscarboxyethyl-5(6)-carboxyfluorescein (BCECF) in isolated myocytes

Isolated rat heart myocytes were loaded with both the Ca2+ sensitive fluorescent probe fura-2/AM and the fluorescent pH indicator 2,7-biscarboxyethyl-5(6)-carboxyfluorescein (BCECF/AM). Changes in [Ca2+]i and pHi were measured simultaneously using digitized video fluorescence microscopy. In measurement of [Ca2+]i and pHi, the ratios of dual-loaded cells were not different from single-loaded cells. Using this method, [Ca2+]i and pHi in myocytes were 48 +/- 7 nM and 7.17 +/- 0.05. It is concluded that [Ca2+]i and pHi could be measured simultaneously in isolated myocyte using dual-loading of fura-2 and BCECF.     

External and internal flavonoids from Madagascarian Uncarina species (Pedaliaceae)

External and internal flavonoids were isolated from 12 Uncarina taxa (Pedaliaceae), endemic to Madagascar. Four flavone aglycones, tricetin 7,3′,5′-trimethyl ether, tricetin 7,4′,5′-trimethyl ether, 5,3′-dihydroxy-6,7,4′,5′-tetramethoxyflavone and eupatorin were isolated from leaf wax of seven Uncarina taxa, Uncarina grandidieri, Uncarina decaryi, Uncarina abbreviata, Uncarina turicana, Uncarina platycarpa, Uncarina leandrii var. leandrii and Uncarina peltata, but not Uncarina stellulifera, Uncarina perrieri, Uncarina sakalava, Uncarina leptocarpa and U. leandrii var. rechbergeri. Furthermore, eight flavonoid glycosides were isolated from the leaves. Major glycosides were apigenin and luteolin 7-O-glucuronides and occurred in all the Uncarina taxa examined, except the absence of the former compound in U. peltata. Other glycosides were identified as hispidulin, jaceosidin, chrysoeriol and tricin 7-O-glucuronides, and luteolin 7,4′-di-O-glucuronide and a flavonol, isorhamnetin 3-O-diglucoside. From the results described above, methylated flavone aglycones and glucuronides were chemical characters of the leaves of Uncarina species, and also may be those of the family Pedaliaceae. Besides, an anthocyanin, two flavonols and three flavones were isolated from the flowers of U. grandidieri, and identified as cyanidin 3-O-rutinoside (anthocyanin), quercetin and isorhamnetin 7-O-glucuronides (flavonols) and apigenin, luteolin and jaceosidin 7-O-glucuronides (flavones).     

An enzyme combination assay for serum sphingomyelin: Improved specificity through avoiding the interference with lysophosphatidylcholine

Serum sphingomyelin (SM) has predictive value in the development of atherosclerosis. Furthermore, SM plays important roles in cell membrane structure, signal transduction pathways, and lipid raft formation. A convenient enzymatic method for SM is available for routine laboratory practice, but the enzyme specificity is not sufficient because of nonspecific reactions with lysophosphatidylcholine (LPC). Based on the differential specificity of selected enzymes toward choline-containing phospholipids, a two-step assay for measuring SM was constructed and its performance was evaluated using sera from healthy individuals on a Hitachi 7170 autoanalyzer. Results from this assay were highly correlated with theoretical serum SM concentrations estimated by subtracting phosphatidylcholine (PC) and LPC concentrations from that of total phospholipids determined using previously established methods. There was a good correlation between the results of SM assayed by the proposed method and the existing enzymatic method in sera from healthy individuals. Moreover, the proposed method was superior to the existing method in preventing nonspecific reactions with LPC present in sera. The proposed method does not require any pretreatment, uses 2.5 μl of serum samples, and requires only 10 min on an autoanalyzer. This high-throughput method can measure serum SM with sufficient specificity for clinical purposes and is applicable in routine laboratory practice.     

Ionotropic glutamate receptor AMPA 1 is associated with ovulation rate

Ionotropic glutamate receptors mediate most excitatory neurotransmission in the central nervous system by opening ion channels upon the binding of glutamate. Despite the essential roles of glutamate in the control of reproduction and anterior pituitary hormone secretion, there is a limited understanding of how glutamate receptors control ovulation. Here we reveal the function of the ionotropic glutamate receptor AMPA-1 (GRIA1) in ovulation. Based on a genome-wide association study in Bos taurus, we found that ovulation rate is influenced by a variation in the N-terminal leucine/isoleucine/valine-binding protein (LIVBP) domain of GRIA1, in which serine is replaced by asparagine. GRIA1(Asn) has a weaker affinity to glutamate than GRIA1(Ser), both in Xenopus oocytes and in the membrane fraction of bovine brain. This single amino acid substitution leads to the decreased release of gonadotropin-releasing hormone (GnRH) in immortalized hypothalamic GT1-7 cells. Cows with GRIA1(Asn) have a slower luteinizing hormone (LH) surge than cows with GRIA1(Ser). In addition, cows with GRIA1(Asn) possess fewer immature ovarian follicles before superovulation and have a lower response to hormone treatment than cows with GRIA1(Ser). Our work identified that GRIA1 is a critical mediator of ovulation and that GRIA1 might be a useful target for reproductive therapy.     

New methods for species and sex determination in three sympatric Mustelids, Mustela itatsi, Mustela sibirica and Martes melampus

We developed novel species and sex determination methods for three Japanese mustelid species. We used DDX3Y to determine sex and generated a primer set to amplify both DDX3X and DDX3Y DNA in Mustela itatsi, M. sibirica and Martes melampus. To determine species and sex simultaneously, we generated fluorescence-labelled primers that give different fragment lengths at D-loop, DDX3X and DDX3Y of these three species using a DNA sequencer.