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Apremilast, an oral small molecule inhibitor of phosphodiesterase 4 (PDE4), is in development for chronic inflammatory disorders, and has shown efficacy in psoriasis, psoriatic arthropathies, and Behçet's syndrome. In March 2014, the US Food and Drug Administration approved apremilast for the treatment of adult patients with active psoriatic arthritis. The properties of apremilast were evaluated to determine its specificity, effects on intracellular signaling, gene and protein expression, and in vivo pharmacology using models of innate and adaptive immunity. Apremilast inhibited PDE4 isoforms from all four sub-families (A1A, B1, B2, C1, and D2), with IC50 values in the range of 10 to 100 nM. Apremilast did not significantly inhibit other PDEs, kinases, enzymes, or receptors. While both apremilast and thalidomide share a phthalimide ring structure, apremilast lacks the glutarimide ring and thus fails to bind to cereblon, the target of thalidomide action. In monocytes and T cells, apremilast elevated intracellular cAMP and induced phosphorylation of the protein kinase A substrates CREB and activating transcription factor-1 while inhibiting NF-κB transcriptional activity, resulting in both up- and down-regulation of several genes induced via TLR4. Apremilast reduced interferon-α production by plasmacytoid dendritic cells and inhibited T-cell cytokine production, but had little effect on B-cell immunoglobulin secretion. In a transgenic T-cell and B-cell transfer murine model, apremilast (5mg/kg/day p.o.) did not affect clonal expansion of either T or B cells and had little or no effect on their expression of activation markers. The effect of apremilast on innate immunity was tested in the ferret lung neutrophilia model, which allows monitoring of the known PDE4 inhibitor gastrointestinal side effects (nausea and vomiting). Apremilast significantly inhibited lung neutrophilia at 1mg/kg, but did not induce significant emetic reflexes at doses <30 mg/kg. Overall, the pharmacological effects of apremilast are consistent with those of a targeted PDE4 inhibitor, with selective effects on innate immune responses and a wide therapeutic index compared to its gastrointestinal side effects., (Copyright © 2014. Published by Elsevier Inc.)

Thalidomide and the immunomodulatory drug, lenalidomide, are therapeutically active in hematological malignancies. The ubiquitously expressed E3 ligase protein cereblon (CRBN) has been identified as the primary teratogenic target of thalidomide. Our studies demonstrate that thalidomide, lenalidomide and another immunomodulatory drug, pomalidomide, bound endogenous CRBN and recombinant CRBN-DNA damage binding protein-1 (DDB1) complexes. CRBN mediated antiproliferative activities of lenalidomide and pomalidomide in myeloma cells, as well as lenalidomide- and pomalidomide-induced cytokine production in T cells. Lenalidomide and pomalidomide inhibited autoubiquitination of CRBN in HEK293T cells expressing thalidomide-binding competent wild-type CRBN, but not thalidomide-binding defective CRBN(YW/AA). Overexpression of CRBN wild-type protein, but not CRBN(YW/AA) mutant protein, in KMS12 myeloma cells, amplified pomalidomide-mediated reductions in c-myc and IRF4 expression and increases in p21(WAF-1) expression. Long-term selection for lenalidomide resistance in H929 myeloma cell lines was accompanied by a reduction in CRBN, while in DF15R myeloma cells resistant to both pomalidomide and lenalidomide, CRBN protein was undetectable. Our biophysical, biochemical and gene silencing studies show that CRBN is a proximate, therapeutically important molecular target of lenalidomide and pomalidomide.

The prostaglandin D(2) receptor type 2 (DP2) and its ligand, PGD(2), have been implicated in the development of asthma and other inflammatory diseases. The authors evaluated the pharmacodynamics, pharmacokinetics and safety of [2'-(3-benzyl-1-ethyl-ureidomethyl)-6-methoxy-4'-trifluoromethyl-biphenyl-3-yl]-acetic acid sodium salt (AM211), a novel and potent DP2 antagonist, in healthy participants. Single and multiple doses of AM211 demonstrated dose-dependent inhibition of eosinophil shape change in blood with near-complete inhibition observed at trough after dosing 200 mg once daily for 7 days. Maximum plasma concentrations and exposures of AM211 increased in a greater-than-dose-proportional manner after single and multiple dosing. After multiple dosing, the exposures on day 7 were higher than on day 1 with accumulation ratio values ranging from 1.4 to 1.5. Mean terminal half-life values ranged from 14 to 25 hours across the dose range of 100 to 600 mg. AM211 was well tolerated at all doses in both the single- and multiple-dose cohorts. These data support additional clinical studies to evaluate AM211 in asthma and other inflammatory diseases.

Lysophosphatidic acid (LPA) is a bioactive phospholipid that signals through a family of at least six G protein-coupled receptors designated LPA₁₋₆. LPA type 1 receptor (LPA₁) exhibits widespread tissue distribution and regulates a variety of physiological and pathological cellular functions. Here, we evaluated the in vitro pharmacology, pharmacokinetic, and pharmacodynamic properties of the LPA₁-selective antagonist AM095 (sodium, {4'-[3-methyl-4-((R)-1-phenyl-ethoxycarbonylamino)-isoxazol-5-yl]-biphenyl-4-yl}-acetate) and assessed the effects of AM095 in rodent models of lung and kidney fibrosis and dermal wound healing. In vitro, AM095 was a potent LPA₁ receptor antagonist because it inhibited GTPγS binding to Chinese hamster ovary (CHO) cell membranes overexpressing recombinant human or mouse LPA₁ with IC₅₀ values of 0.98 and 0.73 μM, respectively, and exhibited no LPA₁ agonism. In functional assays, AM095 inhibited LPA-driven chemotaxis of CHO cells overexpressing mouse LPA₁ (IC₅₀= 778 nM) and human A2058 melanoma cells (IC₅₀ = 233 nM). In vivo, we demonstrated that AM095: 1) had high oral bioavailability and a moderate half-life and was well tolerated at the doses tested in rats and dogs after oral and intravenous dosing, 2) dose-dependently reduced LPA-stimulated histamine release, 3) attenuated bleomycin-induced increases in collagen, protein, and inflammatory cell infiltration in bronchalveolar lavage fluid, and 4) decreased kidney fibrosis in a mouse unilateral ureteral obstruction model. Despite its antifibrotic activity, AM095 had no effect on normal wound healing after incisional and excisional wounding in rats. These data demonstrate that AM095 is an LPA₁ receptor antagonist with good oral exposure and antifibrotic activity in rodent models.

Background and Purpose: The aim of this study was to assess the potential of an antagonist selective for the lysophosphatidic acid receptor, LPA(1), in treating lung fibrosis We evaluated the in vitro and in vivo pharmacological properties of the high affinity, selective, oral LPA(1)-antagonist (4'-{4-[(R)-1-(2-chloro-phenyl)-ethoxycarbonylamino]-3-methyl-isoxazol-5-yl}-biphenyl-4-yl)-acetic acid (AM966)., Experimental Approach: The potency and selectivity of AM966 for LPA(1) receptors was determined in vitro by calcium flux and cell chemotaxis assays using recombinant and native cell cultures. The in vivo efficacy of AM966 to reduce tissue injury, vascular leakage, inflammation and fibrosis was assessed at several time points in the mouse bleomycin model., Key Results: AM966 was a potent antagonist of LPA(1) receptors, with selectivity for this receptor over the other LPA receptors. In vitro, AM966 inhibited LPA-stimulated intracellular calcium release (IC(50)= 17 nM) from Chinese hamster ovary cells stably expressing human LPA(1) receptors and inhibited LPA-induced chemotaxis (IC(50)= 181 nM) of human IMR-90 lung fibroblasts expressing LPA(1) receptors. AM966 demonstrated a good pharmacokinetic profile following oral dosing in mice. In the mouse, AM966 reduced lung injury, vascular leakage, inflammation and fibrosis at multiple time points following intratracheal bleomycin instillation. AM966 also decreased lactate dehydrogenase activity and tissue inhibitor of metalloproteinase-1, transforming growth factor beta1, hyaluronan and matrix metalloproteinase-7, in bronchoalveolar lavage fluid., Conclusions and Implications: These findings demonstrate that AM966 is a potent, selective, orally bioavailable LPA(1) receptor antagonist that may be beneficial in treating lung injury and fibrosis, as well as other diseases that are characterized by pathological inflammation, oedema and fibrosis.

The 5-lipoxygenase-activating protein (FLAP) gene and an increase in leukotriene (LT) production are linked to the risk of asthma, myocardial infarction, and stroke. We evaluated the pharmacodynamics, pharmacokinetics, and tolerability of 3-[3-tert-butylsulfanyl-1-[4-(6-methoxy-pyridin-3-yl)-benzyl]-5-(pyridin-2-ylmethoxy)-1H-indol-2-yl]-2,2-dimethyl-propionic acid (AM103), a novel FLAP inhibitor, in healthy subjects. Single and multiple doses of AM103 demonstrated dose-dependent inhibition of blood LTB(4) production and dose-related inhibition of urinary LTE(4). After a single oral dose (50-1,000 mg) of AM103, the maximum concentration (C(max)) and area under the curve (AUC) in plasma increased in a dose-dependent manner. After multiple-dose administration (50-1,000 mg once daily for 11 days), there were no significant differences in the pharmacokinetic parameters between the first and last days of treatment. AM103 was well tolerated at all doses in both the single- and multiple-dose cohorts. Further clinical trials with AM103 in inflammatory diseases are warranted.

Prior to the discovery of cyclooxygenase-2 (COX-2), a beneficial association was shown between chronic usage of non steroidal anti-inflammatory drugs (NSAIDs), that non-selectively inhibit both cyclooxygenase-1 (COX-1) and COX-2, and prevention of colorectal cancer. The cloning of COX-2 allowed the development of enzyme inhibitors that selectively inhibit COX-2 and also facilitated the expression profiling of COX-2 in many cancer tissues. COX-2 selective inhibitors have shown efficacy in vitro and in vivo in several animal cancer models and in limited human clinical trials. The potency of COX-2 inhibitors in vivo can be attributed to the inhibition of the enzyme in the tumor as well as in stromal cells, resulting in reduction of carcinogen production, anti-proliferative and pro-apoptopic actions within the tumor and anti-angiogenic and pro-immune surveillance activities in endothelial and myeloid cells. The combination of COX-2 inhibitor with standard cancer chemotherapeutic and/or radiation may provide additional therapeutic paradigms in the treatment of various human cancers.

Background: Cysteinyl leukotrienes (CysLTs) are bioactive lipids that have been shown to contribute to allergic and inflammatory diseases. Eosinophils and mast cells have the capacity to produce large amounts of CysLTs after allergic or non-allergic stimulation. Molecular identification of both the synthetic and signalling proteins in the CysLT pathway allows the investigation of expression of the CysLT enzymes and receptors in active allergic rhinitis., Objective: We examined the expression of the proteins involved in the synthesis of CysLTs and the cysteinyl leukotriene-1 (CysLT1) and cysteinyl leukotriene-2 (CysLT2) receptors in inflammatory cells from patients with active seasonal allergic rhinitis., Methods: Nasal lavage samples were obtained from patients during active seasonal allergic rhinitis. Specific cellular immunocytochemical techniques were used to detect the cysteinyl leukotriene synthetic proteins, namely 5-lipoxygenase (5-LO), 5-lipoxygenase-activating protein (FLAP) and leukotriene C4 synthase (LTC4S). In situ hybridization and immunocytochemical techniques were used to identify the mRNA and proteins for the CysLT1 and CysLT2 receptors., Results: 5-LO, FLAP and LTC4S, and the CysLT1 and CysLT2 receptors were expressed in the majority of eosinophils and in subsets of mast cells and mononuclear cells. 5-LO, FLAP and the CysLT1 receptor, but not LTC4S or the CysLT2 receptor, were expressed in a subset of nasal neutrophils., Conclusions: Our study demonstrates the presence of CysLT pathway proteins in key allergic and inflammatory cells from the upper airway of patients with active seasonal allergic rhinitis. Our expression data highlight the potential of CysLT-modifying agents to treat both upper and lower airway symptoms in patients suffering from allergic rhinitis and asthma.

We report the identification, cloning and tissue distributions of ten novel human genes encoding G protein-coupled receptors (GPCRs) GPR78, GPR80, GPR81, GPR82, GPR93, GPR94, GPR95, GPR101, GPR102, GPR103 and a pseudogene, psi GPR79. Each novel orphan GPCR (oGPCR) gene was discovered using customized searches of the GenBank high-throughput genomic sequences database with previously known GPCR-encoding sequences. The expressed genes can now be used in assays to determine endogenous and pharmacological ligands. GPR78 shared highest identity with the oGPCR gene GPR26 (56% identity in the transmembrane (TM) regions). psi GPR79 shared highest sequence identity with the P2Y(2) gene and contained a frame-shift truncating the encoded receptor in TM5, demonstrating a pseudogene. GPR80 shared highest identity with the P2Y(1) gene (45% in the TM regions), while GPR81, GPR82 and GPR93 shared TM identities with the oGPCR genes HM74 (70%), GPR17 (30%) and P2Y(5) (40%), respectively. Two other novel GPCR genes, GPR94 and GPR95, encoded a subfamily with the genes encoding the UDP-glucose and P2Y(12) receptors (sharing >50% identities in the TM regions). GPR101 demonstrated only distant identities with other GPCR genes and GPR102 shared identities with GPR57, GPR58 and PNR (35-42% in the TM regions). GPR103 shared identities with the neuropeptide FF 2, neuropeptide Y2 and galanin GalR1 receptors (34-38% in the TM regions). Northern analyses revealed GPR78 mRNA expression in the pituitary and placenta and GPR81 expression in the pituitary. A search of the GenBank databases with the GPR82 sequence retrieved an identical sequence in an expressed sequence tag (EST) partially encoding GPR82 from human colonic tissue. The GPR93 sequence retrieved an identical, human EST sequence from human primary tonsil B-cells and an EST partially encoding mouse GPR93 from small intestinal tissue. GPR94 was expressed in the frontal cortex, caudate putamen and thalamus of brain while GPR95 was expressed in the human prostate and rat stomach and fetal tissues. GPR101 revealed mRNA transcripts in caudate putamen and hypothalamus. GPR103 mRNA signals were detected in the cortex, pituitary, thalamus, hypothalamus, basal forebrain, midbrain and pons.

Epidemiological studies suggest that nonsteroidal anti-inflammatory agents decrease the risk of colorectal cancer. This is believed to be mediated, at least in part, by inhibition of cyclooxygenase (COX) activity. There are two COX isoenzymes, namely the constitutively expressed COX-1 and the inducible COX-2. COX-2 is overexpressed in adenomas and colorectal cancers, and COX-2-specific inhibitors have been shown to inhibit intestinal polyps in Apc(Delta716) mice more effectively than dual COX-1/COX-2 inhibitors such as sulindac. Various Apc knockout mice, including the multiple intestinal neoplasia (Min) mouse and the Apc(Delta716) mouse, are limited by their lack of large numbers of colonic adenomas and aberrant crypt foci, the putative precursors of large-bowel polyps and cancers. Our DNA mismatch-repair-deficient Min mouse model (Apc+/-Msh2-/-) has genetic features of both familial adenomatous polyposis and hereditary nonpolyposis colorectal cancer, and most importantly, rapidly develops numerous small- and large-bowel adenomas, as well as colonic aberrant crypt foci. The purpose of this study was to determine the effects of COX inhibitors on intestinal adenomas and colonic aberrant crypt foci in this accelerated polyposis, mismatch-repair-deficient Min mouse model, in addition to a standard Min mouse model. Weanling Apc+/-Msh2-/- and Min mice were fed diets containing no drug, sulindac, or a specific COX-2 inhibitor (MF-tricyclic). Apc+/-Msh2-/- and Min mice were sacrificed after 4 weeks and 5 months on diet, respectively. Apc+/-Msh2-/- mice treated with MF-tricyclic had significantly fewer small-bowel polyps (mean +/- SD, 178 +/- 29) compared with mice on sulindac (278 +/- 80), or control diet (341 +/- 43; P < 0.001). There was no difference in numbers of large-bowel polyps or aberrant crypt foci in mice in the three groups. MF-tricyclic was also effective in reducing both small- and large-bowel polyps in Min mice. Western analysis demonstrated COX-2 expression in both large- and small-bowel polyps from mice of both genotypes. This study demonstrates that a specific COX-2 inhibitor is effective in preventing small-bowel polyps in mismatch-repair-deficient Min mice and both small- and large-bowel polyps in standard Min mice. Therefore, specific COX-2 inhibitors may be useful as chemopreventive and therapeutic agents in humans at risk for colorectal neoplasia.

The purpose of this article is to demonstrate the viability of using a high-resolution 3-D head phantom in Monte Carlo N-Particle (MCNP) for boron neutron capture therapy (BNCT) structure dosimetry. This work describes a high-resolution voxel-based model of a human head and its use for calculating absorbed doses to the structures of the brain. The Zubal head phantom is a 3-D model of a human head that can be displayed and manipulated on a computer. Several changes were made to the original head phantom which now contains over 29 critical structures of the brain and head. The modified phantom is a 85 x 109 x 120 lattice of voxels, where each voxel is 2.2 x 2.2 x 1.4 mm3. This model was translated into MCNP lattice format. As a proof of principle study, two MCNP absorbed dose calculations were made (left and right lateral irradiations) using a uniformly distributed neutron disk source with an 1/E energy spectrum. Additionally, the results of these two calculations were combined to estimate the absorbed doses from a bilateral irradiation. Radiobiologically equivalent (RBE) doses were calculated for all structures and were normalized to 12.8 Gy-Eq. For a left lateral irradiation, the left motor cortex receives the limiting RBE dose. For a bilateral irradiation, the insula cortices receive the limiting dose. Among the nonencephalic structures, the parotid glands receive RBE doses that were within 15% of the limiting dose.

The aims of the study are to develop a non-invasive animal model of circular motion exercise and to evaluate the effect of this type of exercise on bone turnover in young rats. The circular motion exercise simulates isometric exercise using an orbital shaker that oscillates at a frequency of 50 Hz and is capable of speeds from 0-400 rpm. A cage is fixed on top of the shaker and the animals are placed inside. When the shaker is turned on, the oscillatory movement should encourage the animals to hold on to the cage and use various muscle forces to stabilize themselves. Rats at 8 weeks of age were trained on the shaker for 6 weeks and static and dynamic histomorphometric analyses were performed for the proximal tibial metaphysis and the tibial shaft. The exercise resulted in no significant effect on animal body weight, gastrocnemius muscle weight and femoral weight. Although the bone formation rate of cancellous and cortical periosteum was increased by the exercise, trabecular bone volume was decreased. The exercise increased periosteal and marrow perimeters and the cross-sectional diameter of cortical bone from medial to lateral without a significant increase in the cortical bone area. These results suggest that circular motion exercise under force without movement or additional weight loading will cause bone-modeling drift with an increase in bone turnover to reconstruct bone shape in adaptation to the demand in strength. Since there is no additional weight loading during circular motion exercise, the net mass of bone is not increased. The bone mass lost in trabecular bone could possibly be due to a re-distribution of mineral to the cortical bone.

Mutations in the human adenomatous polyposis (APC) gene are causative for familial adenomatous polyposis (FAP), a rare condition in which numerous colonic polyps arise during puberty and, if left untreated, lead to colon cancer. The APC gene is a tumor suppressor that has been termed the "gatekeeper gene" for colon cancer. In addition to the 100% mutation rate in FAP patients, the APC gene is mutated in >80% of sporadic colon and intestinal cancers. The Apc gene in mice has been mutated either by chemical carcinogenesis, resulting in the Min mouse Apcdelta850, or by heterologous recombination, resulting in the Apcdelta716 or Apedelta1368 mice (M. Oshima et al., Proc. Natl. Acad. Sci. USA, 92: 4482-4486, 1995). Although homozygote Apc-/- mice are embryonically lethal, the heterozygotes are viable but develop numerous intestinal polyps with loss of Apc heterozygosity within the polyps (M. Oshima et al., Proc. Natl. Acad. Sci. USA, 92: 4482-4486, 1995). The proinflammatory, prooncogenic protein cyclooxygenase (COX)-2 has been shown to be markedly induced in the Apcdelta716 polyps at an early stage of polyp development (M. Oshima et al., Cell, 87: 803-809, 1996). We demonstrate here that treatment with the specific COX-2 inhibitor rofecoxib results in a dose-dependent reduction in the number and size of intestinal and colonic polyps in the Apcdelta716 mouse. The plasma concentration of rofecoxib that resulted in a 55% inhibition of polyp number and an 80% inhibition of polyps > 1 mm in size is comparable with the human clinical steady-state concentration of 25 mg rofecoxib (Vioxx) taken once daily (A. Porras et al., Clin. Pharm. Ther., 67: 137, 2000). Polyps from both untreated and rofecoxib- or sulindac-treated Apcdelta716 mice expressed COX-1 and -2, whereas normal epithelium from all mice expressed COX-1 but minimal amounts of COX-2. Polyps from either rofecoxib- or sulindac-treated mice had lower rates of DNA replication, expressed less proangiogenic vascular endothelial-derived growth factor and more membrane-bound beta-catenin, but showed unchanged nuclear localization of this transcription factor. This study showing the inhibition of polyposis in the Apcdelta716 mouse suggests that the specific COX-2 inhibitor rofecoxib (Vioxx) has potential as a chemopreventive agent in human intestinal and colon cancer.

The majority of genes encoding G protein-coupled receptors were isolated by methods based on sequence similarities found throughout this family. Experimental techniques have exploited these similarities (including low-stringency hybridization, polymerase chain reaction and electronic database searching) to identify genes encoding many pharmacologically recognized receptors and their subtypes. Homology-based searches have revealed receptors for which the endogenous ligands were unknown and these were named orphan receptors. Many orphan receptors are expressed in the brain, suggesting the existence of unidentified neurotransmitters. Methods used to identify ligands for these orphan receptors resulted in the identification of novel ligands and succeeded in pairing previously identified ligands with their receptors. Similar successful strategies are required to characterize the physiological and pathological importance of the remaining orphan receptors to facilitate the discovery of novel drugs for these systems.

The cysteinyl leukotrienes (CysLTs) are important mediators of human asthma. Pharmacologic and clinical studies show that the CysLTs exert most of their bronchoconstrictive and proinflammatory effects through activation of a putative, 7-transmembrane domain, G-protein-coupled receptor, the CysLT1 receptor. The initial molecular characterization of the CysLT1 receptor showed by in situ hybridization, the presence of CysLT1 receptor messenger RNA (mRNA) in human lung smooth-muscle cells and lung macrophages. We confirmed the results of these in situ hybridization analyses for the CysLT1 receptor, and produced the first immunohistochemical characterization of the CysLT1 receptor protein in human lung. The identification of the CysLT1 receptor in the lung is consistent with the antibronchoconstrictive and antiinflammatory actions of CysLT1 receptor antagonists. We also report the expression of CysLT1 receptor mRNA and protein in most peripheral blood eosinophils and pregranulocytic CD34+ cells, and in subsets of monocytes and B lymphocytes.

The contractile and inflammatory actions of the cysteinyl leukotrienes (CysLTs), LTC(4), LTD(4), and LTE(4), are thought to be mediated through at least two distinct but related CysLT G protein-coupled receptors. The human CysLT(1) receptor has been recently cloned and characterized. We describe here the cloning and characterization of the second cysteinyl leukotriene receptor, CysLT(2), a 346-amino acid protein with 38% amino acid identity to the CysLT(1) receptor. The recombinant human CysLT(2) receptor was expressed in Xenopus oocytes and HEK293T cells and shown to couple to elevation of intracellular calcium when activated by LTC(4), LTD(4), or LTE(4). Analyses of radiolabeled LTD(4) binding to the recombinant CysLT(2) receptor demonstrated high affinity binding and a rank order of potency for competition of LTC(4) = LTD(4) LTE(4). In contrast to the dual CysLT(1)/CysLT(2) antagonist, BAY u9773, the CysLT(1) receptor-selective antagonists MK-571, montelukast (Singulair(TM)), zafirlukast (Accolate(TM)), and pranlukast (Onon(TM)) exhibited low potency in competition for LTD(4) binding and as antagonists of CysLT(2) receptor signaling. CysLT(2) receptor mRNA was detected in lung macrophages and airway smooth muscle, cardiac Purkinje cells, adrenal medulla cells, peripheral blood leukocytes, and brain, and the receptor gene was mapped to chromosome 13q14, a region linked to atopic asthma.

Neuromedin U (NMU) is a neuropeptide with potent activity on smooth muscle which was isolated first from porcine spinal cord and later from other species. It is widely distributed in the gut and central nervous system. Peripheral activities of NMU include stimulation of smooth muscle, increase of blood pressure, alteration of ion transport in the gut, control of local blood flow and regulation of adrenocortical function. An NMU receptor has not been molecularly identified. Here we show that the previously described orphan G-protein-coupled receptor FM-3 (ref. 15) and a newly discovered one (FM-4) are cognate receptors for NMU. FM-3, designated NMU1R, is abundantly expressed in peripheral tissues whereas FM-4, designated NMU2R, is expressed in specific regions of the brain. NMU is expressed in the ventromedial hypothalamus in the rat brain, and its level is significantly reduced following fasting. Intracerebroventricular administration of NMU markedly suppresses food intake in rats. These findings provide a molecular basis for the biochemical activities of NMU and may indicate that NMU is involved in the central control of feeding.

In a previous work, we demonstrated that the osteoprogenitors derived from the marrow stroma of the hypophysectomized (HX) rat demonstrate enhanced proliferative and differentiation capacities when placed in an optimal microenvironment. In this study, we sought to investigate the potential of the trabecular osteoblast-like cells of the HX rat. These cells represent a more mature pool of osteoblasts than the progenitors derived from the marrow stroma. We examined all three stages of osteoblast development using trabecular osteoblast-like cells derived from age-matched intact rats as a control. Using thymidine incorporation and cell number as indicators of proliferation, we found that these cells, like the osteoprogenitors derived from the HX rat, demonstrate augmented proliferation when placed in culture. Additionally, type I collagen expression remained at significant levels past the end stages of proliferation, at which point it is expected to be downregulated. Matrix maturation markers, such as alkaline phosphatase activity and bone sialoprotein expression, however, were significantly lower than in the controls. Mineralization potential, as measured by mineralized nodule formation, Ca(2+) content, and OPN and OCN expression, was also significantly reduced. Our results have uncovered an aberrant model of osteoblast development in which proliferation is deregulated, resulting in a minimal capacity of these cells to develop into fully differentiated mineralizing osteoblasts.

We have studied the novel horizontally polymerized mixed trichloropropyl-trichlorooctadecyl silane bonded phase described by Wirth. These materials can be reproducibly prepared and give very high bonded phase density (>7.5 micromol m(-2)). They show significantly improved alkaline stability and chromatographic selectivity towards PAHs similar to conventional monomeric phases. Study of retention by the Linear Solvation Energy Relationship approach as well as measurement of dead volume and retention of methanol indicate that less mobile phase is sorbed by a horizontally polymerized phase than by conventional phases. Silanophilic interaction of amines are decidedly weaker on a silica modified by horizontal polymerization compared to a conventionally modified phase. In addition, this work provides additional support for the "partition-like" retention mechanism of bonded phase RPLC.

Urotensin II (UII) is a neuropeptide with potent cardiovascular effects. Its sequence is strongly conserved among different species and has structural similarity to somatostatin. No receptor for UII has been molecularly identified from any species so far. GPR14 was cloned as an orphan G protein-coupled receptor with similarity to members of the somatostatin/opioid receptor family. We have now demonstrated that GPR14 is a high affinity receptor for UII and designate it UII-R1a. HEK293 cells and COS-7 cells transfected with rat GPR14 showed strong, dose-dependent calcium mobilization in response to fish, frog, and human UII. Radioligand binding analysis showed high affinity binding of UII to membrane preparations isolated from HEK293 cells stably expressing rat GPR14. In situ hybridization analysis showed that GPR14 was expressed in motor neurons of the spinal cord, smooth muscle cells of the bladder, and muscle cells of the heart. The identification of the first receptor for UII will allow better understanding of the physiological and pharmacological roles of UII., (Copyright 1999 Academic Press.)

The development of a COX-2 inhibitor rofecoxib (MK 966, Vioxx) is described. It is essentially equipotent to indomethacin both in vitro and in vivo but without the ulcerogenic side effect due to COX-1 inhibition.

Within 3 h of ingesting an imbalanced amino acid diet (Imb), rats show attenuated intake, which can be ameliorated by prior administration of the serotonin receptor antagonist tropisetron (Trop). Earlier work in which the dorsomedial hypothalamic nucleus (DMN) was electrolytically lesioned (DMNL) determined that this structure plays a role in the early detection of and subsequent adaptation to Imb. However, that study did not address whether cell bodies in the DMN, fibers of passage, or both were involved in the DMNL response to Imb. In the present investigation in experiment 1, rats were given electrolytic DMNL or a sham operation (Sham). The rats were injected with saline (Sal) or Trop just before introduction of Imb. By 3 h Sal-DMNL rats consumed more Imb than did the Sal-Sham rats; intake was normal by 12 h. Trop enhanced Imb intake, with Trop and DMNL being additive. By day 4 the DMNL rats were eating and gaining weight less than were Sham rats. In experiment 2, DMN cell bodies were destroyed by ibotenic acid (Ibo). Sal-injected Ibo-lesioned and Sham rats showed similar food intake depression on Imb; Trop similarly increased Imb intake in both groups. By day 4 both Ibo-L rats were eating and gaining weight less than were Sham rats. In experiment 3, groups of rats were given knife cuts posterior, lateral, ventral, dorsal, or anterior to the DMN. During the first 3 h of consuming Imb, all cuts except posterior enhanced the intake of Imb. Over the next 24 h the anterior cut group continued to eat more Imb than did the Sham rats. In experiment 4 DMNL rats were given novel diets; the DMNL rats did not display a neophilic response. The data suggest that fiber tracts that pass through the DMN may be involved in the early detection of Imb. DMN cell bodies, or fibers of passage, are not involved in the Trop effect. Finally, DMN cell bodies are necessary for proper long-term adaptation to Imb.

The cysteinyl leukotrienes-leukotriene C4(LTC4), leukotriene D4(LTD4) and leukotriene E4(LTE4)-are important mediators of human bronchial asthma. Pharmacological studies have determined that cysteinyl leukotrienes activate at least two receptors, designated CysLT1 and CysLT2. The CysLT1-selective antagonists, such as montelukast (Singulair), zafirlukast (Accolate) and pranlukast (Onon), are important in the treatment of asthma. Previous biochemical characterization of CysLT1 antagonists and the CysLT1 receptor has been in membrane preparations from tissues enriched for this receptor. Here we report the molecular and pharmacological characterization of the cloned human CysLT1 receptor. We describe the functional activation (calcium mobilization) of this receptor by LTD4 and LTC4, and competition for radiolabelled LTD4 binding to this receptor by the cysteinyl leukotrienes and three structurally distinct classes of CysLT1-receptor antagonists. We detected CysLT1-receptor messenger RNA in spleen, peripheral blood leukocytes and lung. In normal human lung, expression of the CysLT1-receptor mRNA was confined to smooth muscle cells and tissue macrophages. Finally, we mapped the human CysLT1-receptor gene to the X chromosome.

G protein-coupled receptors are commonly thought to bind their cognate ligands and elicit functional responses primarily as monomeric receptors. In studying the recombinant gamma-aminobutyric acid, type B (GABAB) receptor (gb1a) and a GABAB-like orphan receptor (gb2), we observed that both receptors are functionally inactive when expressed individually in multiple heterologous systems. Characterization of the tissue distribution of each of the receptors by in situ hybridization histochemistry in rat brain revealed co-localization of gb1 and gb2 transcripts in many brain regions, suggesting the hypothesis that gb1 and gb2 may interact in vivo. In three established functional systems (inwardly rectifying K+ channel currents in Xenopus oocytes, melanophore pigment aggregation, and direct cAMP measurements in HEK-293 cells), GABA mediated a functional response in cells coexpressing gb1a and gb2 but not in cells expressing either receptor individually. This GABA activity could be blocked with the GABAB receptor antagonist CGP71872. In COS-7 cells coexpressing gb1a and gb2 receptors, co-immunoprecipitation of gb1a and gb2 receptors was demonstrated, indicating that gb1a and gb2 act as subunits in the formation of a functional GABAB receptor.

Conditions such as estrogen deficiency, skeletal unloading, and aging have all been demonstrated to have various effects on the proliferation and differentiation of bone marrow stroma-derived osteoprogenitor cells. Here we have sought to examine the effects of pituitary hormone deficiency on the proliferation and the differentiation of these osteoprogenitor cells using the hypophysectomized (HX) rat as a model. In the present study, we use an in vitro culture system to examine the effects of HX on the osteogenic potential of rat bone marrow stroma. With the intact animal as a control, we used [3H]thymidine incorporation and cell number as indexes of proliferation. We also measured alkaline phosphatase enzyme activity, relative levels of osteocalcin expression with RT-PCR, and osteopontin and bone sialoprotein steady-state levels by Northern blot to delineate the effect on differentiation. Our results indicate that osteoprogenitor cells exposed to a pituitary hormone-deficient environment in vivo demonstrate an enhanced proliferative capacity and also exhibit an augmented expression of differentiation markers when exposed to an optimal environment in vitro.

Unlabelled: Many radiopharmaceuticals are excreted from the body through the gastrointestinal (GI) tract. The doses to the walls of the organs involved often are very significant. As significant fractions of the administered activity pass through them, these organs may receive the highest doses in the body for many radiopharmaceuticals. The absorbed dose to these walled organs, from activity in their contents, is typically calculated as 50% of the average absorbed dose to the contents, for nonpenetrating emissions. The internal surface of the GI tract, and to a certain extent the urinary bladder, is lined with a variable thickness of mucus. In addition, the radiosensitive cell populations (crypt or stem cells) are located at some depth into the mucosa. These two factors suggest that the surface dose, often used to characterize the clinically relevant absorbed doses for walled organs, may represent an overestimate in some cases., Methods: In this study, the radiation transport code MCNP was used to simulate the deposition of energy from nonpenetrating emissions of several radionuclides of interest: 90Y, 99mTc,123I and 131I. Absorbed doses as a function of distance from the wall-contents interface were calculated for three geometric shapes representing different organs along the routes of excretion., Results: The absorbed dose from nonpenetrating emissions to the sensitive cell populations was consistently lower than estimated by the standard model assumption. The simulated absorbed doses to radiosensitive cells in the GI tract for 99mTc and 123I are tenfold lower; those for 131I are fivefold lower and those for 90Y are 20% lower., Conclusion: This study demonstrates that the normally reported dose to the walls of hollow organs probably should be modified to account for the attenuation of these nonpenetrating emissions in the linings of the walls. This study also demonstrates that Monte Carlo codes continue to be useful in the evaluation of the dose to sensitive cells in walled organs.

A case study of a young deaf child's conversation development is used to explore the relationship between mode of inquiry and findings resulting from the use of qualitative research methodology. Data were collected through participant observation, videotaping, and interviews in the participants' natural settings. The breadth of findings from thematic analysis of descriptive data and discourse analysis of language samples led to the discovery of the child's language competencies in a field where research usually focuses on language deficits. Additionally, data revealed the contextual features that contributed to the child's conversation development. Scope of findings and implications for educators and researchers provide evidence of the value of qualitative methodology as a mode of inquiry in the field of education of the Deaf.

Within 3 h of ingesting an imbalanced amino acid diet (IAAD), rats show attenuated intake. The associated conditioned taste aversion can be ameliorated by giving the serotonin3 receptor blocker, tropisetron (TROP). A recent c-fos study indicated that the dorsomedial hypothalamic nucleus (DMN) may be activated 2-3 h after ingestion of IAAD. In Experiment 1, DMN-lesioned rats (DMNL) or sham-operated (SHAM) rats were injected with saline (SAL) or TROP just before introduction of IAAD. By 3 h, SAL-DMNL rats consumed more (P < 0.01) of the IAAD than did the SAL-SHAM rats. Thereafter, over the next 21 h, the intake of the SAL-DMNL group returned to control levels. TROP treatment enhanced the intake of the treated groups; the TROP and the lesion effect were additive (P < 0.01). By d 4 of receiving the IAAD, the DMNL groups were eating less than SHAM rats (P < 0.05). The data suggest that the DMN may be involved in the early detection of the amino acid deficiency induced by IAAD, is not involved in the TROP effect and is necessary for proper long-term adaptation to an IAAD.

Leukotriene A4 (LTA4) hydrolase catalyzes the conversion of the unstable epoxide LTA4 [5(S)-trans-5,6-oxido-11,14-cis-eicosatetraenoic acid] into proinflammatory LTB4. During the process of catalyzing this reaction, the enzyme is suicide inactivated by its substrate. In addition, LTA3, and analogue of LTA4 that lacks the C14-C15 double bond, is a potent suicide inhibitor of LTA4 hydrolase. We have synthesized [3H]LTA3 and used this ligand to demonstrate that LTA3 can covalently label LTA4 hydrolase and that this labeling is specifically competed for by bestatin and LTA4. Incubation of recombinant human LTA4 hydrolase with LTA3 followed by proteolysis (endoproteinase Lys-C) resulted in a peptide map with a single modified peptide defining the location of the LTA3 covalent attachment region. This modified 21-amino-acid peptide had a UV absorption spectrum corresponding to a conjugated triene chromophore which established conservation of this structural unit after covalent interaction of LTA3 with LTA4 hydrolase. MALDI-TOF mass spectrometric analysis of the 21-amino-acid peptide adduct revealed an abundant MH+ at m/z 2658, consistent with the predicted nominal mass of the sequenced peptide with the addition of a single LTA3 moiety. Proteolysis of LTA4 hydrolase modified with LTA3 was performed sequentially with endo-Asp-N and endo-Lys-C. The resulting peptide isolated by reverse-phase high-performance liquid chromatography was analyzed by mass spectroscopy revealing two related peptides, D371-K385 (m/z 2018.0) and D375-K385 (m/z 1577.8), both of which retained the elements of LTA3. Postsource decay of m/z 1577.8 resulted in an abundant ion at m/z 536 and an ion of lesser abundance at m/z 856 consistent with cleavage between V381 and P382 that supported assignment of the modified tyrosine residue at Y383. These results suggest nucleophilic attack of a tyrosine residue (Y383) at the conjugated triene epoxide of LTA3 resulting in a triene ether carbinol covalent adduct.

Two cyclooxygenase isozymes catalyze conversion of arachidonic acid to prostaglandin H2: constitutive COX-1 and inducible COX-2. To assess the role of COX-2 in colorectal tumorigenisis, we determined the effects of COX-2 gene (Ptgs2) knockouts and a novel COX-2 inhibitor on Apc delta716 knockout mice, a model of human familial adenomatous polyposis. A Ptgs2 null mutation reduced the number and size of the intestinal polyps dramatically. Furthermore, treating Apc delta716 mice with a novel COX-2 inhibitor reduced the polyp number more significantly than with sulindac, which inhibits both isoenzymes. These results provide direct genetic evidence that COX-2 plays a key role in tumorigenesis and indicate that COX-2-selective inhibitors can be a novel class of therapeutic agents for colorectal polyposis and cancer.

Protecting the facility personnel and the general public from radiation exposure is a primary safety concern of an accelerator-based epithermal neutron irradiation facility. This work makes an attempt at answering the questions "How much?" and "What kind?" of shielding will meet the occupational limits of such a facility. Shielding effectiveness is compared for ordinary and barytes concretes in combination with and without borated polyethylene. A calculational model was developed of a treatment room , patient "scatterer," and the epithermal neutron beam. The Monte Carlo code, MCNP, was used to compute the total effective dose equivalent rates at specific points of interest outside of the treatment room. A conservative occupational effective dose rate limit of 0.01 mSv h-1 was the guideline for this study. Conservative Monte Carlo calculations show that constructing the treatment room walls with 1.5 m of ordinary concrete, 1.2 m of barytes concrete, 1.0 m of ordinary concrete preceded by 10 cm of 5% boron-polyethylene, or 0.8 m of barytes concrete preceded by 10 cm of 5% boron-polyethylene will adequately protect facility personnel.

Selective inhibitors of prostaglandin synthase-2 (PGHS-2) possess potent anti-inflammatory, antipyretic, and analgesic properties but demonstrate reduced side-effects (e.g. gastrotoxicity) when compared with nonselective inhibitors of PGHS-1 and -2. We investigated the mechanism of the differential inhibition of human PGHS-1 (hPGHS-1) and -2 (hPGHS-2) in intact cells by nonsteroidal anti-inflammatory drugs (NSAIDs) and examined factors that contribute to the increased potency of PGHS inhibitors observed in intact cells versus cell-free systems. In intact Chinese hamster ovary (CHO) cell lines stably expressing the hPGHS isozymes, both PGHS isoforms exhibited the same affinity for arachidonic acid. Exogenous and endogenous arachidonic acid were used as substrates by both CHO [hPGHS-1] and CHO [hPGHS-2] cell lines. However, differences were observed in the ability of the hPGHS isoforms to utilize endogenous arachidonic acid released intracellularly following calcium ionophore stimulation or released by human cytosolic phospholipase A2 transiently expressed in the cells. Cell-based screening of PGHS inhibitors demonstrated that the selectivities and potencies of PGHS inhibitors determined using intact cells are affected by substrate concentration and differ from that determined in cell-free microsomal or purified enzyme preparations of PGHS isozymes. The mechanism of inhibition of PGHS isozymes by NSAIDs in intact cells involved difference in their time-dependent inhibition. Indomethacin displayed time-dependent inhibition of cellular hPGHS-1 and -2. In contrast, the selective PGHS-2 inhibitor NS-398 exhibited time-independent inhibition of hPGHS-1 but time-dependent inhibition of hPGHS-2 in intact cells. Reversible inhibition of cellular CHO [hPGHS-1] and CHO [hPGHS-2] was observed with the nonselective NSAIDs ibuprofen and indomethacin, whereas inhibition by the selective PGHS-2 inhibitor DuP-697 was reversible against hPGHS-1 but irreversible against hPGHS-2.

The attachment of an arylacetic or benzoic acid moiety to the thiopyrano[2,3,4-c,d]indole nucleus results in compounds which are highly potent and selective 5-lipoxygenase (5-LO) inhibitors. These compounds are structurally simpler than previous compounds of similar potency in that they contain a single chiral center. From the data presented, 2-[[1-(3-chlorobenzyl)-4-methyl-6-[(5-phenylpyridin-2-yl)methoxy]- 4, 5-dihydro-1H-thiopyrano[2,3,4-c,d]indol-2-yl]methoxy]-phenylacetic acid, 14b, was shown to inhibit 5-hydroperoxyeicosatetraenoic acid (5-HPETE) production by human 5-LO (IC50 of 18 nM). The acid 14b is highly selective as an inhibitor of 5-LO activity when compared to the inhibition of ram seminal vesicle cyclooxygenase (IC50 > 5 microM) or human leukocyte leukotriene A4 (LTA4) hydrolase (IC50 > 20 microM). In addition, 14b was inactive in a 5-lipoxygenase-activating protein (FLAP) binding assay at 10 microM. In vivo studies showed that 14b is bioavailable in rat and functionally active in the hyperreactive rat model of antigen-induced dyspnea (74% inhibition at 0.5 mk/kg po; 2 h pretreatment). In the conscious squirrel monkey model of asthma, 14b showed excellent functional activity at 0.1 mg/kg against antigen-induced bronchoconstriction (94% inhibition of the increase in RL and 100% inhibition in the decrease in Cdyn; n = 4). Resolution of this compound gave (-)-14b, the most potent enantiomer (IC50 = 10 nM in the human 5-LO assay), which was shown to possess the S configuration at the chiral center by X-ray crystallographic analysis of an intermediate. Subsequent studies on the aryl thiopyrano[2,3,4-c,d]indole series of inhibitors led to the discovery of potent dual inhibitors of both FLAP and 5-LO, the most potent of which is 2-[[1-(4-chlorobenzyl)-4-methyl-6-(quinolin-2-ylmethoxy)-4, 5-dihydro-1H-thiopyrano[2,3,4-c,d]indol-2-yl]methoxy]phenylacetic acid, 19. Acid 19 has an IC50 of 100 nM for the inhibition of 5-HPETE production by human 5-LO and is active in a FLAP binding assay with an IC50 of 32 nM. Furthermore, thiopyrano[2,3,4-c,d]indoles such as 1 and 14b are capable of inhibiting the LTC4 synthase reaction in a dose dependent manner (IC50s of 11 and 16 microM, respectively, compared to that of LTC2 at 1.2 microM) in contrast to other, structurally distinct 5-LO inhibitors. It has also been observed that the thiopyrano[2,3,4-c,d]indole class of compounds strongly promotes the translocation of 5-LO from the cytosol to a membrane fraction in the presence or absence of the ionophore A23187.(ABSTRACT TRUNCATED AT 400 WORDS)

In the past, much research on deaf children's language development has been conducted in schools and clinical settings to examine children's achievement of standard English or to study the effectiveness of mode of communication. The purpose of this study was to examine a deaf child's pragmatic development, her abilities as a language user in the naturally occurring situations of her everyday life at home. Observations, videotaping, and interviews were used to collect descriptive data of the child's conversation experiences with hearing family members. Findings reveal that deafness has had an impact on the family's beliefs and communication practices. Data show the child to be a competent communicator as she uses language appropriately for a variety of purposes, demonstrates her knowledge of the structural features of conversation, and employs an array of communication strategies to achieve mutual understanding with family members.

Each of these effector systems has the capacity to initiate airway obstruction or alter airway responsiveness in asthma. It is likely that they act in concert in certain asthmatic settings. Further basic and applied research will define their relative roles in asthma.

The human gene encoding the bifunctional aminopeptidase and epoxide hydrolase enzyme, leukotriene A4 hydrolase (LTA4 hydrolase) has been cloned from a placental lambda phage genomic library. The gene is greater than 35 kbp and contains 19 exons ranging in size over 24-312 bp. The introns range in size over 0.26-5.7 kbp. The essential zinc-binding histidine residues and glutamate residue, which delineate the zinc-binding domain required for both enzyme activities of LTA4 hydrolase, are divided between exons 10 and 11. The LTA4 hydrolase gene was localized to chromosome 12q22 utilizing fluorescence in situ hybridization. Based on the chromosome localization and genomic DNA analysis, LTA4 hydrolase was determined to be a single-copy gene. Primer-extension analysis demonstrated that the transcription initiation site of LTA4 hydrolase mRNA is 151 nucleotides upstream of the initiator ATG. Approximately 4 kbp of 5'-flanking region of the LTA4 hydrolase gene has been obtained and sequencing of 1.4 kb of this 5'-flanking region demonstrated several transcription-factor consensus sequences, including a phorbol-ester-response element (AP2) and two xenobiotic-response elements. The cloning and characterization of the human gene for LTA4 hydrolase provides a basis for further insight into transcriptional regulation of this bifunctional enzyme and its role in various inflammatory processes.

Prostaglandin G/H synthase (PGHS), a key enzyme leading to the formation of prostaglandins, is the target of nonsteroidal antiinflammatory drugs. Two forms of the enzyme have been identified, PGHS-1 and PGHS-2. Epidemiological evidence has suggested that aspirin and other nonsteroidal antiinflammatory drugs may reduce the risk of colorectal cancer. We examined by immunoblot analyses the expression of human PGHS-1 and PGHS-2 protein in 25 matched colon cancer and nontumor tissues, 4 premalignant polyps, 5 control colon tissues from noncancer patients, and 3 matched normal and cancerous breast tissue samples. PGHS-1 was detected in all normal and tumor tissue. In contrast, PGHS-2 was not detected in 23 of 25 normal colon tissues but was detected in 19 of 25 colon tumors. PGHS-2 protein was not observed in four human premalignant polyp samples, control colon from noncancer patients, or matched normal or cancerous breast tissues. These results suggest that the beneficial effects of nonsteroidal antiinflammatory drugs in colon cancer may be mediated by inhibition of PGHS-2.

Transferrin, when injected intracranially into glioblastoma multiforme lesions, acts as a cytotoxic substance. Transferrin, radiolabeled with In-111, can be coinjected and subsequent scintigraphic imaging can demonstrate the biokinetics of the cytotoxic transferrin. The administration of 111In transferrin into a brain tumor results in distribution of radioactivity in the brain, brain tumor, and the cerebrospinal fluid (CSF). Information about absorbed radiation doses to these regions, as well as other nearby tissues and organs, is important for evaluating radiation-related risks from this procedure. The radiation dose is usually estimated for a mathematical representation of the human body. We have included source/target regions for the eye, lens of the eye, spinal column, spinal CSF, cranial CSF, and a 100-g tumor within the brain of an adult male phantom developed by Cristy and Eckerman. The mathematical models of the spinal column, spinal CSF, and the eyes were developed previously, however, these source/targets have not been routinely included in photon transport simulations. Specific absorbed fractions (SAFs) as a function of photon energy were calculated using the ALGAMP computer code, which utilizes Monte Carlo techniques for simulating photon transport. The ALGAMP code was run three times, with the source activity distributed uniformly within the tumor, cranial CSF, and the spinal CSF volumes. These SAFs, which were generated for 12 discrete photon energies ranging from 0.01 to 4.0 MeV, were used with decay scheme data to calculate S-values needed for estimating absorbed doses. S-values for 111In are given for three source regions (brain tumor, cranial CSF, and spinal CSF) and all standard target regions/organs, the eye and lens, as well as to tissues within these source regions. S-values for the skeletal regions containing active marrow are estimated. These results are useful in evaluating the radiation doses from intracranial administration of 111In transferrin. The SAFs are also generally useful for calculation of absorbed dose from any radionuclide in these source regions.

5-Lipoxygenase-activating protein is required for cellular leukotriene synthesis and is the target of the leukotriene biosynthesis inhibitors MK-886 (3-[1-(p-chlorophenyl)-5-isopropyl-3-tert-butylthio-1H- indol-2-yl]-2,2-dimethylpropanoic acid) and MK-591 (3-[1-(4-chlorobenzyl)-3-(t-butylthio)-5-(quinolin-2-ylmethoxy)-indol-2-yl] - 2,2-dimethylpropanoic acid). Recent studies demonstrate that 5-lipoxygenase-activating protein binds arachidonic acid and stimulates the utilization of this substrate by 5-lipoxygenase. The present study utilizes a radioligand binding assay to assess the affinity of 5-lipoxygenase-activating protein for arachidonic acid and the specificity of the fatty acid binding site on 5-lipoxygenase-activating protein. Our findings demonstrate that the presence of a free carboxyl group on fatty acids or leukotriene biosynthesis inhibitors which interact with 5-lipoxygenase-activating protein is not required for specific binding to the protein. However, the degree of saturation significantly affects the affinity of fatty acids for 5-lipoxygenase-activating protein.

In response to calcium ionophore (A23187) stimulation, human granulocyte/macrophage colony-stimulating factor-primed, dimethylsulfoxide-differentiated HL-60 cells (which resemble mature granulocytes) synthesized leukotrienes (LTs) LTA4, LTB4, LTC4, and LTD4. The synthesis of the sulfidopeptide LTs, LTC4 and LTD4, was specifically inhibited in cells incubated in the presence of both A23187 and phorbol-12-myristate-13-acetate (PMA), an activator of protein kinase C (PKC). In contrast, neither the synthesis of LTB4, a product of the nonpeptide branch of the LT pathway, nor the formation of LTA4, the precursor for both branches of the LT biosynthetic pathway, was significantly affected by the presence of PMA during A23187 stimulation. The inhibition by PMA of LTC4 production in A23187-stimulated HL-60 cells was dose dependent, with an IC50 value of approximately 3.5 nM. The PKC inhibitor staurosporine completely reversed the inhibition by PMA of LTC4 production in A23187-stimulated cells, in a dose-dependent fashion, with an IC50 value of approximately 30 nM. Bisindolylmaleimide, another PKC inhibitor, was also able to prevent PMA-mediated inhibition of LTC4 formation, whereas inhibitors of protein kinase A, tyrosine kinases, or the respiratory-burst oxidase were not. Measurement of LTC4 synthase enzymatic activity in cells challenged with A23187 and PMA in the presence or absence of staurosporine demonstrated that the activity of the LTC4 synthase enzyme was inhibited in cells costimulated with A23187 and PMA and that inhibition could also be completely prevented by the presence of staurosporine. Because PMA is known to activate PKC, and staurosporine and bisindolylmaleimide are inhibitors of PKC, these results suggest that LTC4 synthase in HL-60 cells may be phosphoregulated.

The intracellular distribution of the enzyme 5-lipoxygenase (5-LO) and 5-lipoxygenase-activating protein (FLAP) in resting and ionophore-activated human leukocytes has been determined using immuno-electronmicroscopic labeling of ultrathin frozen sections and subcellular fractionation techniques. 5-LO is a 78-kD protein that catalyzes the conversion of arachidonic acid to leukotrienes. FLAP is an 18-kD membrane bound protein that is essential for leukotriene synthesis in cells. In response to ionophore stimulation, 5-LO translocates from a soluble to a sedimentable fraction of cell homogenates. In activated leukocytes, both FLAP and 5-LO were localized in the lumen of the nuclear envelope. Neither protein could be detected in any other cell compartment or along the plasma membrane. In resting cells, the FLAP distribution was identical to that observed in activated cells. In addition, subcellular fractionation techniques showed > 83% of immunoblot-detectable FLAP protein and approximately 64% of the FLAP ligand binding activity was found in the nuclear membrane fraction. A fractionation control demonstrated that a plasma membrane marker, detected by a monoclonal antibody PMN13F6, was not detectable in the nuclear membrane fraction. In contrast to FLAP, 5-LO in resting cells could not be visualized along the nuclear envelope. Except for weak labeling of the euchromatin region of the nucleus, 5-LO could not be readily detected in any other cellular compartment. These results demonstrate that the nuclear envelope is the intracellular site at which 5-LO and FLAP act to metabolize arachidonic acid, and that ionophore activation of neutrophils and monocytes results in the translocation of 5-LO from a nonsedimentable location to the nuclear envelope.

High-level expression of human leukotriene (LT) A4 hydrolase has been established in Sf9 insect cells using the recombinant baculovirus system. LTA4 hydrolase activity in this system is at least 50-fold higher than previously achieved in a bacterial cell system. Recombinant viral human LTA4 hydrolase (rvHLTA4h) was used for coinfection studies with recombinant viral 5-lipoxygenase (rvH5LO). When Sf9 cells expressing 5-lipoxygenase are incubated in the presence of A23187 and arachidonic acid, (5S)-hydroperoxy-6-trans-8,11,14-cis-eicosatetraenoic acid 5-H(P)ETE and LTA4 are synthesized in a ratio of 5:1 for 5-H(P)ETE/LT. Coexpression of 5-lipoxygenase and LTA4 hydrolase in these insect cells results in the synthesis of 5-H(P)ETE, LTA4 and in addition LTB4, and the ratio shifts to 2:1 for 5-H(P)ETE/LT. The production of enzymically formed LTB4 after addition of arachidonic acid to the Sf9 cells coinfected with LTA4 hydrolase and 5-lipoxygenase is the first demonstration of channeling of arachidonic acid to LTB4 in an engineered intact cell system. This delineates a novel biological system to synthesize significant amounts of the potent chemotactic agent, LTB4. Studies in which Sf9 cells infected with rvH5LO were incubated with Sf9 cells infected with rvHLTA4h resulted in export of LTA4 from the rvH5LO cells and transcellular metabolism of LTA4 to LTB4 in the rvHLTA4h Sf9cells.

The evolution of MK-0591 (3-[1-(4-chlorobenzyl)-3-(t-butylthio)-5-(quinolin-2-ylmethoxy+ ++)indol-2-yl]- 2,2-dimethylpropanoic acid), 12, a potent, orally active leukotriene biosynthesis inhibitor is described. MK-0591 is currently undergoing clinical evaluation as a potential agent for the treatment of asthma and inflammatory bowel disease. It acts through a novel mechanism by a specific interaction with a membrane protein, 5-lipoxygenase activating protein (FLAP), which has been shown to be essential for LT synthesis in inflammatory cells. A brief comparison of its biological activity with that of its progenitors MK-886 and L-674,636 is described.

In a previous study, osteosarcoma cells expressing both 5-lipoxygenase (5-LO) and 5 lipoxygenase-activating protein (FLAP) synthesized leukotrienes upon A23187 stimulation (Dixon, R. A. F., R. E. Diehl, E. Opas, E. Rands, P. J. Vickers, J. F. Evans, J. W. Gillard, and D. K. Miller. 1990. Nature (Lond.). 343:282-284). Osteosarcoma cells expressing 5-LO but not expressing FLAP were unable to synthesize leukotrienes. Thus, it was determined that FLAP was required for the cellular synthesis of leukotrienes. To examine the role of FLAP in A23187-induced translocation of 5-LO to a membrane fraction, we have studied the A23187-stimulated translocation of 5-LO in osteosarcoma cells expressing both 5-LO and FLAP, and in osteosarcoma cells expressing 5-LO only. We demonstrate that in cells expressing both 5-LO and FLAP, 5-LO translocates to membranes in response to A23187 stimulation. This 5-LO translocation is inhibited when cells are stimulated in the presence of MK-886. In osteosarcoma cells expressing 5-LO but not expressing FLAP, 5-LO is able to associate with membranes following A23187 stimulation. In contrast to the cells containing both 5-LO and FLAP, MK-886 is unable to prevent 5-LO membrane association in cells transfected with 5-LO alone. Therefore, we have demonstrated that in this cell system, 5-LO membrane association and activation can be separated into at least two distinct steps: (1) calcium-dependent movement of 5-LO to membranes without product formation, which can occur in the absence of FLAP (membrane association), and (2) activation of 5-LO with product formation, which is FLAP dependent and inhibited by MK-886 (enzyme activation).

The earliest reported case of fetal supraventricular tachycardia at 17 weeks' gestation causing hydrops fetalis is presented. Maternal treatment with digoxin and procainamide successfully cardioverted the fetus with resolution of the hydrops. Using this combination, sinus rhythm was maintained until term.

5-Lipoxygenase-activating protein (FLAP) is specifically labeled by [125I]L-669,083 and [125I]L-691,678, photoaffinity analogues of two classes of potent leukotriene biosynthesis inhibitors. Because human FLAP contains only a single tryptophan residue at position 72 and two internal methionine residues at positions 89 and 125, we have used reagents that specifically cleave at these residues, in conjunction with antipeptide antisera, to localize the site of attachment of the photoaffinity ligands. Immunoprecipitation of specifically labeled peptide fragments after digestion of photoaffinity-labeled FLAP by iodosobenzoic acid at 72Trp demonstrates that the inhibitors bind to FLAP amino-terminal to this residue. This finding is consistent with similar immunoprecipitation studies after digestion at methionine residues using cyanogen bromide. These findings localize the site of attachment of the inhibitors to a region of FLAP that includes the hydrophilic loop between the proposed first and second transmembrane regions. Based on these findings, site-directed mutagenesis of human FLAP was performed to define key amino acids involved in inhibitor binding. Using a radioligand binding assay, analysis of mutants of human FLAP expressed in COS-7 cells demonstrates that a number of residues in the amino-terminal half of the first hydrophilic loop of the protein can be deleted without significantly affecting inhibitor binding. In contrast, no inhibitor binding was detectable with mutants in which amino acid residues in the carboxyl-terminal half of this loop were deleted. Furthermore, a point mutation of 62Asp to asparagine results in a mutant with dramatically reduced affinity for inhibitors. This loss of affinity was not displayed by a mutant in which 62Asp was mutated to a glutamate residue, suggesting that a negative charge associated with residue 62 may be critical for inhibitor binding. The roles that amino acid residues in the carboxyl-terminal half of the first hydrophilic loop of FLAP may play in the binding of leukotriene biosynthesis inhibitors are currently under investigation.

MK-0591 (3-[1-(4-chlorobenzyl)-3-(t-butylthio)-5-(quinolin-2-yl-methoxy)- indol-2-yl]-2,2-dimethyl propanoic acid, previously L-686,708) is a potent inhibitor of leukotriene (LT) biosynthesis in intact human and elicited rat polymorphonuclear leukocytes (PMNLs) (IC50 values 3.1 and 6.1 nM, respectively) and in human, squirrel monkey, and rat whole blood (IC50 values 510, 69, and 9 nM, respectively). MK-0591 had no effect on rat 5-lipoxygenase. MK-0591 has a high affinity for 5-lipoxygenase activating protein (FLAP) as evidenced by an IC50 value of 1.6 nM in a FLAP binding assay and inhibition of the photoaffinity labelling of FLAP by two different photoaffinity ligands. Inhibition of activation of 5-lipoxygenase was shown through inhibition of the translocation of the enzyme from the cytosol to the membrane in human PMNLs. MK-0591 was a potent inhibitor of LT biosynthesis in vivo, first, following ex vivo challenge of blood obtained from treated rats and squirrel monkeys, second, in a rat pleurisy model, and, third, as monitored by inhibition of the urinary excretion of LTE4 in antigen-challenged allergic sheep. Inhibition of antigen-induced bronchoconstriction by MK-0591 was observed in inbred rats pretreated with methysergide, Ascaris-challenged squirrel monkeys, and Ascaris-challenged sheep (early and late phase response). These results indicate that MK-0591 is a potent inhibitor of LT biosynthesis both in vitro and in vivo indicating that the compound will be suitable for assessing the role of leukotrienes in pathological situations.

A binding assay has been developed to measure the affinity of leukotriene synthesis inhibitors for 5-lipoxygenase-activating protein (FLAP), using human leukocyte membranes as the source of FLAP and a radioiodinated leukotriene synthesis inhibitor, 125I-L-691,831, as ligand. Linearity of specific binding of radiolabeled ligand was demonstrated with increasing protein and ligand concentrations. Saturation analysis of radioligand binding showed a Kd of 6 nM and a Bmax that, depending on the membrane preparation, varied between 8 and 53 pmol/mg of protein. An excellent correlation was shown between affinity for FLAP in the binding assay and inhibition of leukotriene synthesis in human polymorphonuclear leukocytes for compounds from two structurally distinct classes, namely indoles and quinolines. A large number of membrane-active compounds did not compete with 125I-L-691,831 binding to FLAP. In addition, direct 5-lipoxygenase inhibitors and a selection of eicosanoids were unable to compete for FLAP binding. This study validates a selective binding assay for leukotriene synthesis inhibitors whose protein target is FLAP.

Four mouse monoclonal IgG1 antibody-producing cell lines (5LO-1, 5LO-2, 5LO-3, 5LO-4), produced against highly purified human leukocyte 5-lipoxygenase have been characterized. The monoclonal antibodies produced by these cell lines exhibited differential reactivity against 5-lipoxygenase as determined by ELISA and immunoprecipitation analyses. Monoclonal antibodies 5LO-2 and 5LO-3 inhibited the activity of recombinant human leukocyte 5-lipoxygenase in a dose-dependent manner. This inhibition was selective for 5-lipoxygenase activity since these monoclonal antibodies did not inhibit human leukocyte 15-lipoxygenase or porcine leukocyte 12-lipoxygenase.