Leukotriene B 4 ((5S,12R)-dihydroxy-6,14-cis-8,10-trans-eicosatetraenoic acid (LTB 4 )) 1 is a metabolite of arachidonic acid and is one of the most potent activators of granulocytes and macrophages (1-3). BLT, the LTB 4 -specific G-protein-coupled receptor (GPCR), is a target for anti-inflammatory drugs, and many BLT antagonists have been developed and are being evaluated. No BLT antagonists are yet approved available for clinical use; their lack of efficacy may be due in part to the presence of the other LTB 4 receptors. We cloned BLT1, a high affinity LTB 4 receptor (4), and BLT2, a low affinity LTB 4 receptor (5), and showed that BLT1 is expressed almost exclusively in peripheral leukocytes, whereas BLT2 is expressed ubiquitously with the highest expression in spleen. The structural similarities of these receptors (45% amino acid identity) and the low homologies of BLTs to other known GPCRs suggest that these BLTs comprise a novel receptor family. BLT1 and BLT2 form a gene cluster both in human and mouse genomes.The human BLT2 open reading frame overlaps the promoter region of BLT1 (6), suggesting the expression of these two LTB 4 receptors is tightly intertwined. Human granulocytes, eosinophils, and mononuclear cells express both BLT1 and BLT2 (7), so the precise pharmacological characterization of these two receptors using native cells is difficult. In this paper, we report the inhibitory effects of various BLT antagonists and eicosanoids on LTB 4 binding using CHO cells stably expressing either human BLT1 or BLT2. We also show that several hydroxyeicosanoids other than LTB 4 bind to and activate BLT2 but not BLT1. These findings provide insights into the possible functions of BLT2 and information helpful for the isolation of the other related GPCRs that recognize eicosanoids. (8) and all of the eicosanoids other than LTB 4 were purchased from Cayman Chemical Co. LTB 4 is a generous gift from Ono Pharmaceutical Co. (Osaka, Japan). LY 255283 (9) and LY 223982 (10) were from Lilly Research Laboratories. CP 105696 (11) and CP 195543 (12) are from Pfizer Inc. ZK 158252 is from Schering AG (Berlin, Germany). [ 3 H]LTB 4 (6956 GBq/mmol) was purchased from PerkinElmer Life Sciences. EXPERIMENTAL PROCEDURES Materials-U75302Cell Culture and Flow Cytometry-CHO cells stably expressing FLAG-tagged human BLT1 (CHO-FLAG-BLT1) or hemagglutinin (HA)-tagged human BLT2 (CHO-HA-BLT2) were established as described previously (5, 13). The cells were cloned by limiting dilution and their expression of the receptors confirmed by staining the cells with antibodies against the epitope added to the amino terminus of the receptors. The cells were fixed with PBS(Ϫ) containing 0.5% (w/v) paraformaldehyde for 5 min on ice and blocked with PBS(Ϫ) containing 2% goat serum (Life Technologies, Inc.). The cells were incubated with 30 g/ml anti-FLAG antibody (clone M5, Eastman Kodak), 10 g/ml anti-HA antibody (clone CA12-5, Roche Molecular Biochemicals), or 30 g/ml control mouse IgG (Santa Cruz Biotechnology) in PBS(Ϫ) containing 2% oat seru...

Leukotriene B4 (LTB4) is a potent chemoattractant and activator of both granulocytes and macrophages. The actions of LTB4 appear to be mediated by a specific G protein–coupled receptor (GPCR) BLT1, originally termed BLT (Yokomizo, T., T. Izumi, K. Chang, Y. Takuwa, and T. Shimizu. 1997. Nature. 387:620–624). Here, we report the molecular cloning of a novel GPCR for LTB4, designated BLT2, which binds LTB4 with a Kd value of 23 nM compared with 1.1 nM for BLT1, but still efficiently transduces intracellular signaling. BLT2 is highly homologous to BLT1, with an amino acid identity of 45.2%, and its open reading frame is located in the promoter region of the BLT1 gene. BLT2 is expressed ubiquitously, in contrast to BLT1, which is expressed predominantly in leukocytes. Chinese hamster ovary cells expressing BLT2 exhibit LTB4-induced chemotaxis, calcium mobilization, and pertussis toxin–insensitive inhibition of adenylyl cyclase. Several BLT1 antagonists, including U 75302, failed to inhibit LTB4 binding to BLT2. Thus, BLT2 is a pharmacologically distinct receptor for LTB4, and may mediate cellular functions in tissues other than leukocytes. BLT2 provides a novel target for antiinflammatory therapy and promises to expand our knowledge of LTB4 function. The location of the gene suggests shared transcriptional regulation of these two receptors.

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This paper is a study of comparisons between five types of 100 MW Very Large‐Scale Photovoltaic Power Generation (VLS‐PV) Systems, from economic and environmental viewpoints. The authors designed VLS‐PV systems using typical PV modules of multi‐crystalline silicon (12·8% efficiency), high efficiency multi‐crystalline silicon (15·8%), amorphous silicon (6·9%), cadmium tellurium (9·0%), and copper indium selenium (11·0%), and evaluated them by Life‐Cycle Analysis (LCA). Cost, energy requirement, and CO2 emissions were calculated. In addition, the authors evaluated generation cost, energy payback time (EPT), and CO2 emission rates. As a result, it was found that the EPT is 1·5–2·5 years and the CO2 emission rate is 9–16 g‐C/kWh. The generation cost was 11–12 US Cent/kWh on using 2 USD/W PV modules, and 19–20 US Cent/kWh on using 4 USD/W PV module price. Copyright © 2007 John Wiley & Sons, Ltd.

Leukotriene B4 (LTB4) is a lipid mediator that activates leukocytes and is involved in host defense and inflammation. BLT1, a high-affinity receptor for LTB4 (originally termed BLT), is expressed exclusively in inflammatory cells and is inducible in macrophages upon activation. The mechanisms of tissue-specific expression and induction of BLT1 are important for the understanding of mechanism of onset and the potential treatment of inflammatory disorders. Here, we report the genomic structure and a promoter analysis of the human BLT1 gene, with an emphasis on the mechanism of cell-specific transcription. No TATA or CAAT elements exist around the transcription initiation sites, but a GC-rich sequence is observed in this region. A reporter gene assay revealed that a region ∼80 basepair upstream from the initiator sequence is required for the basal transcription of the BLT1 gene. Sp1 was found to be a major activator of basal transcription by electrophoretic mobility shift assays and site-directed mutagenesis. The CpG sites of the BLT1 promoter region were highly methylated in BLT1-nonexpressing cells, but not methylated in BLT1-expressing cells. Further, methylation of this region in vitro inhibited the promoter activity to ∼15% of the control. Thus, methylation at CpG sites in the promoter region is important for cell-specific transcription of the BLT1 gene. The promoter region of the BLT1 gene is localized within the open reading frame (ORF) of the BLT2 gene, which encodes a low-affinity receptor for LTB4 (Yokomizo, T., K. Kato, K. Terawaki, T. Izumi, and T. Shimizu. 2000. J. Exp. Med. 192:421–431). To our knowledge, this is the first example of “promoter in ORF” in higher eukaryotes.

Tebipenem pivoxil (TBPM-PI) is an oral carbapenem antibiotic for treating otolaryngologic and respiratory infections in pediatric patients. This agent is a prodrug to improve intestinal absorption of TBPM, an active form, and an absorption rate of TBPM-PI is higher than those of other prodrug-type β-lactam antibiotics. In the present study, we hypothesized that a certain mechanism other than simple diffusion is involved in the process of improved intestinal absorption of TBPM-PI and examined the mechanism. TBPM-PI uptake by Caco-2 cells was decreased by ATP-depletion and lowering the temperature to 4 °C, suggesting the contribution of carrier-mediated transport mechanisms. This uptake was partially decreased by ACE inhibitors, and the reduction of the absorption by captopril was observed by in vivo study and in situ single-pass intestinal perfusion study in rat, supporting the contribution of influx transporters. Since some ACE inhibitors and β-lactam antibiotics are reported to be substrates of PEPT and OATP families, we measured transporting activity of TBPM-PI by intestinally expressed transporters, PEPT1, OATP1A2, and OATP2B1. As a result, significant transport activities were observed by both OATP1A2 and OATP2B1 but not by PEPT1. Interestingly, pH dependence of TBPM-PI transports was different between OATP1A2 and OATP2B1, showing highest activity by OATP1A2 at pH 6.5, while OATP2B1-mediated uptake was higher at neutral and weak alkaline pH. OATP1A2 exhibited higher affinity for TBPM-PI (K(m) = 41.1 μM) than OATP2B1 (K(m) > 1 mM) for this agent. These results suggested that TBPM-PI has high intestinal apical membrane permeability due to plural intestinal transport routes, including the uptake transporters such as OATP1A2 and OATP2B1 as well as simple diffusion.

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