Identification of the intracellular region of the leukotriene B4 receptor type 1 that is specifically involved in Gi activation

Many G-protein-coupled receptors can activate more than one G-protein subfamily member. Leukotriene B4 receptor type 1 (BLT1) is a high affinity G-protein-coupled receptors for leukotriene B4 functioning in host defense, inflammation, and immunity. Previous studies have shown that BLT1 utilizes different G-proteins (the Gi family and G16 G-proteins) in mediating diverse cellular events and that truncation of the cytoplasmic tail of BLT1 does not impair activation of Gi and G16 proteins. To determine responsive regions of BLT1 for G-protein coupling, we performed an extensive mutagenesis study of its intracellular loops. Three intracellular loops (i1, i2, and i3) of BLT1 were found to be important for both Gi and G16 coupling, as judged by Gi-dependent guanosine 5'-(gamma-thio) triphosphate (GTPgammaS) binding and G16-dependent inositol phosphate accumulation assays. The i3-1 mutant, with a mutation at the i3 amino terminus, exhibited greatly reduced GTPgammaS binding but intact inositol phosphate accumulation triggered by leukotriene B4 stimulation. These results suggest that the i3-1 region is required only for Gi activation. Moreover, in the i3-1 mutant, the deficiency in Gi activation was accompanied by a loss of the high affinity leukotriene B4 binding state seen with the wild type receptor. A three-dimensional model of BLT1 constructed based on the structure of bovine rhodopsin suggests that the i3-1 region may consist of the cytoplasmic end of the transmembrane helix V, which protrudes the helix into the cytoplasm. From mutational studies and three-dimensional modeling, we propose that the extended cytoplasmic helix connected to the transmembrane helix V of BLT1 might be a key region for selective activation of Gi proteins.     

DNA recombination protein-dependent mechanism of homoplasmy and its proposed functions

Homoplasmy is a basic genetic state of mitochondria, in which all of the hundreds to thousands of mitochondrial (mt)DNA copies within a cell or an individual have the same nucleotide-sequence. It was recently found that "vegetative segregation" to generate homoplasmic cells is an active process under genetic control. In the yeast Saccharomyces cerevisiae, the Mhr1 protein which catalyzes a key reaction in mtDNA homologous recombination, plays a pivotal role in vegetative segregation. Conversely, within the nuclear genome, homologous DNA recombination causes genetic diversity. Considering these contradictory roles of this key reaction in DNA recombination, possible functions of homoplasmy are discussed.     

A developed determination of midazolam and 1'-hydroxymidazolam in plasma by liquid chromatography-mass spectrometry: application of human pharmacokinetic study for measurement of CYP3A activity

This paper describes sensitive and reliable determination of midazolam (MDZ) and its major metabolite 1'-hydroxymidazolam (1-OHMDZ) in human plasma by liquid chromatography-mass spectrometry (LC-MS) with a sonic spray ionization (SSI) interface. MDZ, 1-OHMDZ and diazepam as an internal standard were extracted from 1ml of alkalinized plasma using n-hexane-chloroform (70:30, v/v). The extract was injected into an analytical column (YMC-Pak Pro C(18), 50mmx2.0mmi.d.). The mobile phase for separation consisted of 10mM ammonium acetate and methanol (50:50, v/v) and was delivered at a flow-rate of 0.2ml/min. The drift voltage was 100V. The sampling aperture was heated at 120 degrees C and the shield temperature was 260 degrees C. The total time for chromatographic separation was less than 16min. The validated concentration ranges of this method were 0.25-50ng/ml for both MDZ and 1-OHMDZ. Mean recoveries were 93.6% for MDZ and 86.6% for 1-OHMDZ. Intra- and inter-day coefficient variations were less than 6.5 and 5.5% for MDZ, and 6.1 and 5.7% for 1-OHMDZ at 0.3, 4, 20 and 40ng/ml. The limits of quantification were 0.25ng/ml for both MDZ and 1-OHMDZ. This method was sensitive and reliable enough for pharmacokinetic studies on healthy volunteers, and was applied for the measurement of CYP3A activity in humans after an intravenous (1mg) and a single-oral administration (2mg) of subtherapeutic MDZ dose.     

Soy sauce increased the oxidative stress tolerance of nematode via p38 MAPK pathway

Soy sauce – a fermented food made from soybeans and wheat – is considered a healthy seasoning, but little scientific evidence is available to support this. In this study, physiological effects of soy sauce were analyzed using Caenorhabditis elegans. When soy sauce was fed to C. elegans together with Escherichia coli OP50, fat accumulation decreased, and resistance to oxidative stress by H₂O₂ was greatly increased in the nematodes. qRT-PCR revealed that mRNA expression of oxidative stress tolerance genes, including sod, ctl, and gpx, was markedly increased in soy sauce-fed nematodes. Worms ingesting soy sauce showed high mitochondrial membrane potential and reactive oxygen species (ROS) and low intracellular ROS, suggesting that soy sauce induced mitohormesis and decreased cytoplasmic ROS. Therefore, soy sauce ingestion affects the mitochondria and may alter the fat metabolism in C. elegans. Furthermore, the increase in oxidative stress tolerance is mediated through p38 MAPK pathway.     

Discovery and structure-activity relationship of imidazolinylindole derivatives as kallikrein 7 inhibitors

A series of imidazolinylindole derivatives were discovered as novel kallikrein 7 (KLK7, stratum corneum chymotryptic enzyme) inhibitors. Structure-activity relationship (SAR) studies led to the identification of potent human KLK7 inhibitors. By further modification of the benzenesulfonyl moiety to overcome species differences in inhibitory activity, potent inhibitors against both human and mouse KLK7 were identified. Furthermore, the complex structure of 25 with mouse KLK7 could explain the SAR and the cause of the species differences in inhibitory activity.     

Long-term exposure to superantigen induces p27Kip1 and Bcl-2 expression in effector memory CD4+ T cells

The long-term exposure of mice to superantigen SEA using a mini-osmotic pump (SEA pump) induced a long-lasting expansion of Vβ3⁺CD4⁺ T cells with T helper (Th) 2 cell-type properties. Removal of the SEA pump 10 days after pump implantation did not significantly alter the level of Vβ3⁺CD4⁺ T cell expansion/maintenance. Furthermore, CFSE-labeled CD4⁺ T cells failed to divide when transferred to post-implantation day 15 mice. Thus, CD4⁺ T cells appeared to survive for at least 30 days in the absence of a sufficient amount of antigen to trigger cell division. STAT6 deficient mice, in which Th2 cell development is largely impaired, also exhibited a protracted cell expansion, similar to that observed in normal mice, suggesting that the Th2 cell property is dispensable for the maintenance of Vβ3⁺CD4⁺ T cell expansion. The expanded CD4⁺ T cells on post-implantation day 26 were arrested in the G₀/G₁ phase of the cell cycle and showed a lower level of cell division upon restimulation. The Cdk inhibitor p27^Kip1 was highly expressed, and Cdk2 was downregulated. Moreover, the CD4⁺ T cells were resistant to in vitro apoptosis induction in parallel with their level of Bcl-2 expression. Collectively, the Vβ3⁺CD4⁺ T cells appeared to develop into long-lived memory T cells with cell cycle arrest upon long-term exposure to SEA.     

Two X family DNA polymerases, λ and μ, in meiotic tissues of the basidiomycete, Coprinus cinereus

The X family DNA polymerases λ (CcPolλ) and μ (CcPolμ) were shown to be expressed during meiotic prophase in the basidiomycete, Coprinus cinereus. These two polymerases are the only members of the X family in the C. cinereus genome. The open reading frame of CcPolλ encoded a predicted product of 800 amino acid residues and that of CcPolμ of 621 amino acid residues. Both CcPolλ and CcPolμ required Mn²⁺ ions for activity, and both were strongly inhibited by dideoxythymidine triphosphate. Unlike their mammalian counterparts, CcPolλ and CcPolμ had no terminal deoxynucleotidyl transferase activity. Immunostaining analysis revealed that CcPolλ was present at meiotic prophase nuclei in zygotene and pachytene cells, which is the period when homologous chromosomes pair and recombine. CcPolμ was present in a slightly wider range of cell stages, zygotene to diplotene. In analyses using D-loop recombination intermediate substrates, we found that both CcPolλ and CcPolμ could promote primer extension of an invading strand in a D-loop structure. Moreover, both polymerases could fully extend the primer in the D-loop substrate, suggesting that D-loop extension is an activity intrinsic to CcPolλ and CcPolμ. Based on these data, we discuss the possible roles of these polymerases in meiosis.     

Fluorometric immunoassay based on pH-sensitive dye-encapsulating liposomes and gramicidin channels

This article describes a new method for direct fluorometric immunoassay with a liposome array using pH-sensitive dye (BCECF [2',7'-bis(carboxyethyl)-4 or 5-carboxyfluorescein])-encapsulating liposomes immobilized on an avidin slip and gramicidin channels. The liposomes were composed of phosphatidylcholine (PC), cholesterol (Chol), biotinylated phosphatidylethanolamine (B-cap-PE), and recognition sites (1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine-N-(2,4-dinitrophenyl) [DNP-PE], Fab' fragment of anti-substance P, and Fab' of anti-neurokinin A). The addition of gramicidin induced release of H(+) ions from the inner solution (pH 5.5) to the outer one (pH 7.8), enhancing fluorescence of BCECF (1.0mM) encapsulated in liposome. The binding of an analyte (anti-dinitrophenyl [anti-DNP], avidin, substance P, or neurokinin A) to the membrane-bound recognition sites caused further enhancement of fluorescence of BCECF due to a local distortion of the bilayer structure that affects the channel kinetics of gramicidin. The intensity of fluorescence from the immobilized liposomes 60 min after the addition of gramicidin (10 ng/ml) increased with an increase in the concentration of anti-DNP ranging from 1.2 x 10(-8) to 1.2 x 10(-6)g/ml, avidin ranging from 1.0 x 10(-8) to 1.0 x 10(-6)g/ml, substance P ranging from 1.0 x 10(-8) to 1.0 x 10(-6)g/ml, and neurokinin A ranging from 1.0 x 10(-8) to 1.0 x 10(-6)g/ml. The direct fluorometric immunoassay with a liposome array is simple and easy to carry out. The intensity of fluorescence emitted from the immobilized liposomes is directly measured after incubation with a sample solution and a gramicidin solution in sequence without washing steps. The assay allows simultaneous quantification of multiple components without labeling of antibody or antigen with a fluorescent tag. The liposome-based assay is discussed in terms of principle, sensitivity, and selectivity.     

Abnormality in initiation program of DNA replication is monitored by the highly repetitive rRNA gene array on chromosome XII in budding yeast

We have shown previously that perturbation of origin firing in chromosome replication causes DNA lesions and triggers DNA damage checkpoint control, which ensures genomic integrity by stopping cell cycle progression until the lesions are repaired or by inducing cell death if they are not properly repaired. This was based on the observation that the temperature-sensitive phenotype of orc1-4 and orc2-1 mutants required a programmed action of the RAD9-dependent DNA damage checkpoint. Here, we report that DNA lesions in the orc mutants are induced much more quickly and frequently within the rRNA gene (rDNA) locus than at other chromosomal loci upon temperature shift. orc mutant cells with greatly reduced rDNA copy numbers regained the ability to grow at restrictive temperatures, and the checkpoint response after the temperature shift became weak in these cells. In orc2-1 cells, completion of chromosomal duplication was delayed specifically on chromosome XII, where the rDNA array is located, and the delay was partially suppressed when the rDNA copy number was reduced. These results suggest that the rDNA locus primarily signals abnormalities in the initiation program to the DNA damage checkpoint and that the rDNA copy number modulates the sensitivity of this monitoring function.     

Incongruence between mtDNA phylogeny and morphologial and ecological characters in loaches of the genus Lefua (Balitoridae, Cypriniformes)

To elucidate the phylogenetic position of Lefua loaches from Aichi and Shizuoka Prefectures of Honshu Island, Japan, we determined their nucleotide sequences for the mitochondrial D-loop region and compared these to sequences from four other Lefua species: L. costata, L. nikkonis, L. echigonia, and L. sp. Loaches identified as L. sp. on the basis of morphology comprised a clade (the L. sp. Tokai population) that grouped together with L. echigonia; hence, the processes involved in evolution within the genus Lefua were unclear. We performed randomly amplified polymorphic DNA (RAPD) analyses to obtain genetic information on nuclear DNA. The RAPD patterns of the L. sp. Tokai population differed from those of the local L. echigonia and L. sp. populations. The L. sp. Tokai population was similar to L. echigonia with regard to mitochondrial DNA but differed from L. echigonia and L. sp. with respect to nuclear DNA; this indicated that the evolutionary background of the L. sp. Tokai population was unique. We suggest that introgression of mitochondria occurred from L. echigonia to the L. sp. Tokai population, and speculate on the process of evolution of the latter population of Lefua. As with six L. echigonia populations and two L. sp. populations, we regard the L. sp. Tokai population as an evolutionary significant unit (ESU) that qualifies for protection as an endangered loach.