RESUMO
Rapid downregulation of L-selectin expression occurs in response to leukocyte activation, and it has been speculated to be an integral process in the adhesion cascade leading to neutrophil recruitment to sites of inflammation. It has previously been proposed that L-selectin is proteolytically cleaved from the cell surface; however, the nature of the cleavage site has been unknown. We have produced polyclonal antisera against the extracellular domain and against the cytoplasmic domain of L-selectin. Both antisera immunoprecipitate the intact form of L-selectin from metabolically labeled phytohemagglutinin-stimulated lymphoblasts and peripheral blood neutrophils. In addition, the anti-cytoplasmic domain serum, but not the antiectodomain serum, immunoprecipitate a 6-kD species from PMA activated lymphoblasts and formylmethionylleucylphenylalanine-activated neutrophils. Conversely, the antiectodomain serum but not the anti-cytoplasmic domain serum immunoprecipitate a 68-kD soluble form of L-selectin from the supernatant of PMA-activated lymphoblasts. The appearance of the 6-kD species on activated cells correlated with the disappearance of the intact form of L-selectin and the appearance of the soluble form of L-selectin. A third polyclonal serum generated against the membrane proximal region of the ectodomain also reacted with the 6-kD species, indicating that this is a transmembrane peptide of L-selectin. That the 6-kD species is derived from L-selectin was confirmed by immunoprecipitation of the 6-kD species from L-selectin transfectants but not from mock transfectants. Radiochemical sequence analysis defined a cleavage site between Lys321 and Ser322, which would predict a transmembrane fragment consistent in size with the observed 6-kD fragment. A Ser-Phe-Ser motif adjacent to the cleavage site is conserved between human, mouse, and rat L-selectin, and a related motif is found proximal to transmembrane domains of other downregulated proteins, such as ACE, CD16-II, and TNF-RII, suggesting the possibility of a common recognition motif.
Assuntos
Moléculas de Adesão Celular/metabolismo , Glicoproteínas de Membrana/metabolismo , Sequência de Aminoácidos , Animais , Anticorpos/imunologia , Western Blotting , Moléculas de Adesão Celular/química , Moléculas de Adesão Celular/imunologia , Linhagem Celular , Membrana Celular/metabolismo , Humanos , Selectina L , Linfócitos/citologia , Linfócitos/imunologia , Linfócitos/metabolismo , Glicoproteínas de Membrana/química , Glicoproteínas de Membrana/imunologia , Camundongos , Dados de Sequência Molecular , N-Formilmetionina Leucil-Fenilalanina/farmacologia , Neutrófilos/citologia , Neutrófilos/imunologia , Neutrófilos/metabolismo , Fito-Hemaglutininas , Ratos , Homologia de Sequência de Aminoácidos , Acetato de Tetradecanoilforbol , TransfecçãoRESUMO
The zeta chains of the T cell receptor complex play a critical role in the initiation of proximal signaling events upon T cell activation. Three pairs of potential tyrosine phosphorylation sites are located within the cytoplasmic domains of the zeta chains. Subsequent to engagement of the T cell receptor, one or more of these tyrosine residues is phosphorylated. The phosphotyrosine residues, along with flanking amino acids, form an activation motif (and are shared by signaling subunits in the TCR, B cell receptor, and FcgammaRI) termed tyrosine-based activation motifs (ITAMs). ITAMs serve as binding sites for SH2 domain-containing proteins. Recent evidence suggests that the zeta chains provide docking space for several key signal transduction molecules such as ZAP-70, p56lck, and Shc. To determine if ZAP-70, p56lck, and Shc bind to particular zeta chain ITAM sequences, quantitative free-solution measurements of binding affinities (Kd) were obtained by use of surface plasmon resonance technology. The results indicate that binding affinities of distinct SH2 domains to individual and paired phosphorylation sites greatly differ, and may dictate the sequence of signal transduction events.
Assuntos
Proteínas de Membrana/metabolismo , Proteínas Tirosina Quinases/metabolismo , Receptores de Antígenos de Linfócitos T/metabolismo , Domínios de Homologia de src , Quinases da Família src/metabolismo , Sequência de Aminoácidos , Glutationa Transferase/metabolismo , Proteína Tirosina Quinase p56(lck) Linfócito-Específica , Dados de Sequência Molecular , Fosforilação , Proteínas Recombinantes de Fusão/metabolismo , Proteína-Tirosina Quinase ZAP-70RESUMO
Experimental and computational methods were developed for surface plasmon resonance (SPR) measurements involving interactions between a solution-binding component and a surface-immobilized ligand. These protocols were used to distinguish differences in affinity between the SH2 domain of lck and phosphotyrosyl peptides. The surface-immobilized ligand was the phosphotyrosyl peptide EPQpYEEIPIA, which contains a consensus sequence (pYEEI) for binding lck SH2. In the kinetic experiment, SPR phenomena were measured during association and dissociation reactions for a series of glutathione-S-transferase (GST)-SH2 concentrations, generating a set of SPR curves. A global computational analysis using an A + B<==>AB model resulted in single set of parameter estimates and statistics. In an abbreviated format, an equilibrium experiment was designed so that equilibrium constants (Keq) could be determined rapidly and accurately. A competitive equilibrium assay was developed for GST-SH2 in which Keq values for a series of phosphotyrosyl peptides (derived from the pYEEI sequence) varied over 3 orders of magnitude. Interestingly, these results highlighted the significance of the +1 glutamate in providing high-affinity binding to the SH2 domain. For most drug discovery programs, these Keq determinations are a sufficient measure of potency for the primary screen, with koff and kon determined in a secondary assay. Thus, the application of these techniques to SPR binding phenomena should prove valuable in the discovery and design of receptor-ligand antagonists.
Assuntos
Peptídeos/metabolismo , Proteínas Tirosina Quinases/metabolismo , Tirosina/análogos & derivados , Sequência de Aminoácidos , Ligação Competitiva , Cinética , Ligantes , Proteína Tirosina Quinase p56(lck) Linfócito-Específica , Dados de Sequência Molecular , Peptídeos/química , Fosfotirosina , Ligação Proteica , Proteínas Tirosina Quinases/química , Análise Espectral/métodos , Tirosina/química , Tirosina/metabolismoRESUMO
p56lck is a member of the src family of tyrosine kinases. Through modular binding units called SH2 domains, p56lck promotes phosphotyrosine-dependent protein-protein interactions and plays a critical role in signal transduction events that lead to T-cell activation. Starting from the phosphorylated dipeptide (2), a high-affinity ligand for the p56lck SH2 domain, we have designed novel dipeptides that contain monocharged, nonhydrolyzable phosphate group replacements and bind to the protein with KD's in the low micromolar range. Replacement of the phosphate group in phosphotyrosine-containing sequences by a (R/S)-hydroxyacetic (compound 8) or an oxamic acid (compound 10) moiety leads to hydrolytically stable, monocharged ligands, with 83- and 233-fold decreases in potency, respectively. This loss in binding affinity can be partially compensated for by incorporating large lipophilic groups at the inhibitor N-terminus. These groups provide up to 13-fold increases in potency depending on the nature of the phosphate replacement. The discovery of potent (2-3 microM), hydrolytically stable dipeptide derivatives, bearing only two charges at physiological pH, represents a significant step toward the discovery of compounds with cellular activity and the development of novel therapeutics for conditions associated with undesired T-cell proliferation.
Assuntos
Dipeptídeos/síntese química , Proteína Tirosina Quinase p56(lck) Linfócito-Específica/metabolismo , Domínios de Homologia de src , Cristalografia por Raios X , Dipeptídeos/química , Ligantes , Modelos Moleculares , Ligação Proteica , Relação Estrutura-AtividadeRESUMO
Nevirapine, a dipyridodiazepinone, is a highly specific inhibitor of HIV-1 reverse transcriptase (RT) which exhibits an IC50 = 84nM in enzyme assays and IC50 = 40nM against HIV-1 replication in cell culture. This nonnucleoside inhibitor acts noncompetitively with respect to nucleoside triphosphates, template and primer suggesting that nevirapine does not bind to the active site of RT. Studies employing an azido analogue of nevirapine as a photoaffinity probe indicated that one molecule of inhibitor is sufficient to inactivate one molecule of heterodimeric enzyme and demonstrated that only the p66 subunit of p66/p51 heterodimeric RT is covalently labeled by this probe. When subjected to trypic mapping, Tyr 181 and Tyr 188 were labeled with probe and consequently these aromatic residues are apparently near or actually within the RT binding site for nevirapine. The extent to which Tyr 181 and Tyr 188 participate/contribute to nevirapine binding was determined by making amino acid substitutions at these positions using the corresponding residues from HIV-2 RT which is not sensitive to nevirapine. A change at either position dramatically decreased the enzymes' sensitivity to nevirapine, as well as to TIBO derivative and Merck L-693,593, indicating that both Tyr 181 and 188 are crucial for inhibitor-enzyme interaction. Cell culture selection in the continued presence of nevirapine results in the appearance of resistant HIV-1, Tyr 181 to Cys, raising the concern that combination drug therapy will be required in the clinic.
Assuntos
Azepinas/farmacologia , HIV-1/efeitos dos fármacos , Piridinas/farmacologia , Inibidores da Transcriptase Reversa , Sequência de Aminoácidos , Animais , Azepinas/imunologia , Sítios de Ligação , Resistência Microbiana a Medicamentos , Humanos , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida , Nevirapina , Piridinas/imunologia , Relação Estrutura-Atividade , Especificidade por SubstratoRESUMO
Nevirapine (BI-RG-587) is a potent inhibitor of the polymerase activity of reverse transcriptase of human immunodeficiency virus type-1. Nevirapine, as well as several other non-nucleoside compounds of various structural classes, bind strongly at a site which includes tyrosines 181 and 188 of the p66 subunit of reverse transcriptase. The chromatography which was utilized to explore this binding site is described. BI-RH-448 and BI-RJ-70, two tritiated photoaffinity azido analogues of nevirapine, are each crosslinked to reverse transcriptase. The use of several HPLC-based techniques employing different modes of detection makes it possible to demonstrate a dramatic difference between the two azido analogues in crosslinking behavior. In particular, by comparing HPLC tryptic peptide maps of the photoadducts formed between reverse transcriptase and each azido analogue, it can be shown that crosslinking with BI-RJ-70 but not with BI-RH-448 is more localized, stable, and hence exploitable for the identification of the specifically bonded amino acid residue(s). In addition, comparison of the tryptic maps also makes it feasible to assess which rings of the nevirapine structure are proximal or distal to amino acid side chains of reverse transcriptase. Finally, another feature of the HPLC peptide maps is the application of on-line detection by second order derivative UV absorbance spectroscopy to identify the crosslinked amino acid residue.
Assuntos
Marcadores de Afinidade , Cromatografia Líquida de Alta Pressão/métodos , Reagentes de Ligações Cruzadas , HIV-1/enzimologia , Piridinas/metabolismo , DNA Polimerase Dirigida por RNA/metabolismo , Azepinas/metabolismo , Benzodiazepinonas/metabolismo , Transcriptase Reversa do HIV , Humanos , Nevirapina , Mapeamento de Peptídeos , Fotoquímica , Piridinas/farmacologia , Inibidores da Transcriptase Reversa , Análise de Sequência , Espectrofotometria Ultravioleta , TripsinaRESUMO
Fourteen mature anestrous bitches were used to determine the effectiveness of pretreatment with an orally active progestogen to prevent premature luteolysis of induced corpora lutea (CL) in the anestrous bitch. In Group 1, seven bitches were treated orally with megestrol acetate (Ovaban((R))) at the rate of 2.2 mg/kg body weight for eight days. Three days later, the bitches were treated daily with pregnant mare's serum gonadotropin (PMSG) (44 IU/kg body weight) administered intramuscularly for nine consecutive days, and each bitch was given 500 IU human chorionic gonadotropin (HCG) on day 10, or on the first day of induced estrus if the bitches exhibited estrus while being treated with PMSG. A control group (Group 2) of seven bitches was not treated with Ovaban((R)) but was similarly given PMSG and HCG. Estrus was detected twice daily using a vasectomized male dog and verified by vaginal cytology. Blood samples were obtained on the first day of induced estrus (day 0) and every other day until day 90 post-estrus. Plasma progesterone (P(4)) concentrations were determined by a non-extraction solid phase radioimmunoassay (RIA), and data were analyzed by Student's t-test. There was no significant difference between the progesterone profiles of both groups of bitches. In addition, P(4) values were less than 1 ng/ml by day 50 post-estrus. Results of this study suggested that pretreatment with an orally active progestogen was not effective in preventing premature luteolysis of induced CL in the anestrous bitch.
RESUMO
Each of 24 pasture-reared crossbred beef heifers were herded from a large pasture on day 14 of the estrous cycle and assigned randomly to one of four treatment groups as follows: Field Control (FC), Field ACTH (FA), Pen Control (PC) and Pen ACTH (PA). Field groups were maintained in a small field, and pen groups were confined in a pen in a pole barn. ACTH groups received 200 IU of ACTH IM daily for days 17 through 21 of the cycle and control groups received only the gelatin carrier IM on the same cycle days. Average cycle lengths for FA and PA heifers were 25 and 24.3 days with an average period from plasma progesterone decline below 1 ng/ml to estrus of 5.5 days. During the ACTH injection period, follicular growth was suppressed and the proestrus plasma estrogen rise was delayed. Average cycle lengths for FC and PC heifers were 20.8 and 22.8 days respectively. All control group heifers exhibited estrus within 2 days of the plasma progesterone decline below 1 ng/ml. In addition, pen confinement heifers showed a trend for extended luteal function and consequent extended estrous cycle length.
RESUMO
Uterine biopsy in the mare on day 4 post-ovulation causes an acute inflammatory reaction which results in premature luteolysis. In this study, seven mares (4 to 6 years of age) were used in a switchback experimental design to test the hypothesis that in the mare parenterally administered PBZ will block luteolysis induced by uterine biopsy on day 4 post-ovulation. All mares were allowed two normal estrous cycles (range 18 to 24 days). On the first day of estrus of the third estrous cycle each mare was intravenously given 2 grams PBZ (treatment) or 10 ml 0.9% saline (control) daily until signs of estrus were exhibited. The day of ovulation (day 0) was determined by rectal palpation and subsequently verified by peripheral plasma progesterone concentrations. On day 4 following ovulation all mares were subjected to uterine biopsy, and subsequent estrus detection was performed daily using an andro-genized gelding. A total of 19 estrous cycles (ten for PBZ treatment and nine for controls) were evaluated. Mean number of days (+/-SE) from uterine biopsy to induced estrus was 5.00+/-0.16 for control cycles and was significantly different (P<0.025) when compared with 9.20+/-0.34 days for treatment cycles. Results of this study suggest that PBZ can block luteolysis in the mare induced by uterine biopsy on day 4 post-ovulation, possibly as a result of accumulating PBZ in acutely inflamed uterine tissue and inhibiting prostaglandin synthesis.
RESUMO
Results of two experiments are described. In the first experiment, forty-one mixed-breed goats (does) with unknown gestation lengths were given 10 mg prostaglandin F(2alpha) (PGF(2alpha)) (2 doses of 5 mg each, 24 h apart) i.m. Blood samples were obtained before each treatment with PGF(2alpha) by jugular venipuncture, and plasma progesterone (P(4)) concentrations were determined by a nonextraction solid-phase radioimmunoassay. P(4) concentrations (ng/ml) were significantly decreased (15.47 vs 1.55, P<0.005) 24 h after the first injection of PGF(2alpha). A total of 63 fetuses was collected within 46.5 h following the first injection. Mean (+/- SE) crownrump lengths and body weights of 62 fetuses were 21.46 +/- 0.29 centimeters (cm) and 575.00 +/- 20.60 g, respectively. Based on these findings, the mean gestation length of these does was estimated to be 86.96 +/- 0.74 d. Thirty-one does retained their placenta for 12 to 72 h and were treated with a single injection of 5 mg PGF(2alpha) and 800 mg oxytetracycline i.m. Placental expulsion in all does occurred within 24 h posttreatment. The results of this study suggest that two doses of 5 mg PGF(2alpha) intramuscularly (i.m.) 24 h apart is an effective abortifacient at about 3 mo of pregnancy in does. In the second experiment, 38 does from the first experiment were divided in two groups of 19 each on Day 13 postabortion. Group A (treated) was given 50 ug GnRH i.m. while Group B (control) received 1 ml 0.9% saline i.m. Blood samples were obtained prior to treatment and on Day 23 postabortion and assayed for P(4) concentrations. There was no significant difference (P>0.10) in P(4) concentrations of samples obtained pre- and post-GnRH treatment. However, 14 of 19 and 12 of 19 in Groups A and B, respectively, exhibited estrus within 52 days following abortion. Twenty-six does were bred naturally and 17 became pregnant.
RESUMO
Rectal temperatures and hormone concentrations were monitored at intervals of 2 to 3 weeks, and milk, milk fat, and California mastitis test scores at intervals of 1 week in five shaded and in four nonshaded early lactation cows. Measurements were taken from September to December in the mildly heat stressing climate of Oahu, Hawaii. The daily ambient temperature flux ranged from 22 C to 29 C in September to 20 C to 25 C in December. Average daily temperature-humidity index (THI) values were 75 to 70 for September and December, respectively. Average daily THI values were correlated with rectal temperatures in nonshaded cows and were negatively correlated with plasma adrenal cortex hormones (corticoids) in shaded cows, plasma thyroid hormone in shaded and nonshaded cows, and with milk production in nonshaded cows. Estimated milk production decline per unit increase in THI was 0.32 kg. Nonshaded cows had higher rectal temperatures, a trend for lower plasma corticoids, produced less milk and milk fat, and had higher California mastitis test scores. Shaded cows maintained a higher fat percentage at THI above 74. Average plasma thyroid hormone values were not different between treatment groups. Both groups failed to attain normal rectal temperatures at night. Afternoon rectal temperatures were more highly correlated with the rectal temperature with which the cow started the day than they were with the THI of the day itself.
Assuntos
Bovinos/fisiologia , Clima Tropical , Corticosteroides/sangue , Animais , Temperatura Corporal , California , Laticínios , Umidade , Leite/efeitos da radiação , Estações do Ano , Luz Solar , Temperatura , Hormônios Tireóideos/sangueRESUMO
Jugular blood samples were collected prepartum and postpartum from 97 Holstein cows and heifers. Samples were analyzed for total serum cholesterol and plasma glucose. Plasma samples taken 4, 11, 18, and 25 days postpartum were also analyzed for progesterone. Concentrations of cholesterol, glucose, and progesterone were evaluated in relation to summer- and winter-calving seasons, milk production, lactation number, days-to-conception, number of postcalving uterine infusions given, and time relative to calving. A temperature-humidity index was used as a covariate in the analysis to adjust the data for climatic effects so that seasonal effects other than temperature and humidity could be determined. Average plasma glucose was within the normal range (62 +/- 8 mg/dl). It increased before calving and then declined to a minimum value between 11 and 25 days postpartum. Glucose then increased after 25 days for the summer-calving group and remained relatively stable for the winter-calving cows. Blood glucose concentrations were inversely related to milk production. Negative correlations existed between milk production and plasma glucose at days 4, 11, 18, 25, and 39 postpartum. First-lactation heifers had higher blood glucose levels than cows in their second or later lactation. Blood glucose concentrations were not related to days-to-conception over both seasons. Average serum cholesterol was within the normal range (125 +/- 29 mg/dl). It decreased before calving and then increased for 88 days after calving. Summer-calving cows had higher serum cholesterol concentrations prepartum and winter-calving cows had higher concentrations from 39 through 88 days postpartum. Cholesterol concentrations were directly related to milk production from 25 through 88 days postpartum.(ABSTRACT TRUNCATED AT 250 WORDS)
Assuntos
Glicemia/metabolismo , Bovinos/fisiologia , Colesterol/sangue , Fertilidade , Animais , Bovinos/sangue , Feminino , Lactação , Leite/metabolismo , Período Pós-Parto , Gravidez , Estações do AnoRESUMO
Jugular blood samples were obtained from 55 summer-calving and 40 winter-calving Holstein cows fed diets which averaged 7.4 micrograms of Cu/g. Samples were collected within 3 days of 30, 11, and 4 days before calving and 4, 11, 18, 25, 39, 60, 88, 116, and 147 days after calving. Packed cell volume and plasma Cu concentrations were determined. Blood components were analyzed in relationship to season, milk production, lactation number, number of days to conception, number of postcalving uterine infusions, and time relative to calving. The environmental temperature-humidity index was used as a covariate in the analysis, so that seasonal effects represented feeding or production differences between seasons and were not due to the direct effects of climate. Plasma Cu concentrations were within the usual laboratory limits (1.05 +/- 0.20 microgram/ml). These values were unrelated to PCV in the summer-calving cows, but were slightly related in the winter-calving cows (r = 0.11). Plasma Cu values increased around the time of calving, with the maximum level (1.13 microgram/ml) occurring on postpartum day 11. Plasma Cu concentration was higher in cows that conceived within 80 days of calving than in cows which conceived at 120 days or more. Differences in plasma Cu among fertility groups were most evident during postpartum days 25 to 60. However, the relationship between plasma Cu concentrations and fertility was not consistent. Significant interactions existed for season X fertility groups and milk production groups X fertility groups. Plasma Cu concentration was significantly higher for the summer-calving cows and was inversely related to milk production level.(ABSTRACT TRUNCATED AT 250 WORDS)
Assuntos
Bovinos/fisiologia , Cobre/sangue , Fertilidade , Hematócrito/veterinária , Lactação , Animais , Feminino , Leite/metabolismo , Gravidez , Estações do AnoRESUMO
Holstein cows (n = 104) and heifers (n = 35) were used to determine the effects of treatment with selenium (Se) and vitamin E on whole blood Se values and fertility. At 21 +/- 3 days before parturition, 55 cows and 19 heifers were injected with 680 IU of vitamin E and 50 mg of Se as sodium selenite. Treatment had no effect on days to first estrus, days to first service, days to conception, services per conception, or number of uterine infusions required. Blood samples were obtained from 33 nontreated cows and heifers and 32 treated animals at 21 and 14 days (+/- 3) before calving and at 7 and 14 days (+/- 3) after calving. Mean whole blood Se level at -21 days (day 0 was day of parturition) and before Se-vitamin E treatment was 0.109 micrograms/ml. At days -14, 7, and 14, blood Se was significantly higher in the treated than the non-treated animals. Blood Se was lower on all sampling days in cows calving in July through November than in cows calving in December through April. Heifers in their 1st lactation had lower blood Se concentrations than did cows in their 2nd or later lactation. In feeds sampled at 2-week intervals, mean concentrations of Se in Bahia grass, mixed ryegrass and oats, corn silage, and sorghum silage were less than 0.1 micrograms/g on a dry matter basis. Bermuda grass, alfalfa hay, and concentrates contained greater than 0.1 micrograms of Se/g. Large variation existed in Se concentrations of individual feedstuffs.
Assuntos
Ração Animal/análise , Bovinos/fisiologia , Fertilidade/efeitos dos fármacos , Prenhez/efeitos dos fármacos , Selênio/farmacologia , Vitamina E/farmacologia , Animais , Bovinos/sangue , Feminino , Lactação/efeitos dos fármacos , Gravidez , Estações do Ano , Ácido Selenioso , Selênio/análise , Selênio/sangueRESUMO
Metabolic profiles have been used in efforts to predict periparturient problems and fertility, to diagnose metabolic disease, and to assess nutritional status. Results have been varied. Until knowledge and technology provide improved blood constituent panels, the metabolic profile should not be the first step in the diagnostic process. Rather, such profiles should follow an assessment of management practices and an evaluation of diet. However, these profiles may help to confirm the diagnosis, to convince dairy farmers that management changes are desirable, or to monitor improvement in herd animals. At this point, their major contribution has been to increase our understanding of the factors contributing to changes in blood constituent concentrations, which, in turn, has led to more efficient means of diagnosis. Except in cases of gross mismanagement, these profiles do not offer a "quick fix." In many of the reported cases in which diagnosis of herd problems was attributed to the metabolic profile, the clinician should have been able to identify the problem before the profile was conducted. Profiles are to be recommended when the cause of an existing problem is still not identified or resolved after a complete evaluation. The profile may aid in identifying a factor that has been overlooked. Profiles are not for clinicians who do not have an interest in upgrading their understanding of the factors involved, or who do not have a source of knowledgeable advice.
Assuntos
Doenças dos Bovinos/diagnóstico , Doenças Metabólicas/veterinária , Animais , Análise Química do Sangue/veterinária , Bovinos , Doenças dos Bovinos/sangue , Doenças dos Bovinos/metabolismo , Índices de Eritrócitos/veterinária , Doenças Metabólicas/sangue , Doenças Metabólicas/diagnósticoRESUMO
Epoxyeicosanoids, including the epoxyeicosatrienoic acids are signaling molecules which appear to help ameliorate the effects of a wide variety of pathological conditions. The enzyme soluble epoxide hydrolase (sEH) metabolizes these molecules by converting them to their corresponding vicinal diols. Inhibition of sEH either by knockout or chemical inhibitors increases epoxyeicosanoid levels in vivo and provides significant organ protection in models of brain, cardiac, and renal injury. sEH also appears to be involved in modulating inflammation, pain pathways, pulmonary function, hypertension, and diabetes. Potent sEH inhibitors have been developed in academic, pharmaceutical, and biotech laboratories and described in the patent and scientific literature. Most of the inhibitor scaffolds employ a urea or amide which functions as an active-site transition state mimic. Arête Therapeutics compound AR9281 successfully completed phase Ia and 1b studies. A phase IIa proof of concept trial for treatment of impaired glucose tolerance has been completed, but the results are not yet reported.