Structure-activity relationships of 2,4-diphenyl-1H-imidazole analogs as CB2 receptor agonists for the treatment of chronic pain

A series of 2,4-diphenyl-1H-imidazole analogs have been synthesized and displayed potent human CB2 agonist activity. Many of these analogs showed high functional selectivity over human CB1 receptors. The syntheses, structure-activity relationships, and selected pharmacokinetic data of these analogs are described.     

Intrinsic differences in the response of the human lutropin receptor versus the human follitropin receptor to activating mutations

In contrast to the human lutropin receptor (hLHR), very few naturally occurring activating mutations of the structurally related human follitropin receptor (hFSHR) have been identified. The present study was undertaken to determine if one aspect underlying this discrepancy might be a general resistance of the hFSHR to mutation-induced constitutive activity. Five different mutations were introduced into both the hLHR and hFSHR (four based on activating mutations of the hLHR gene, one based on an activating mutation of the hFSHR gene). Our results demonstrate that hFSHR constitutively activating mutants (CAMs) were not as active as hLHR CAMs containing the comparable mutation. Furthermore, although all hFSHR CAMs exhibited strong promiscuous activation by high concentrations of the other glycoprotein hormone receptors, hLHR CAMs showed little or no promiscuous activation. Our in vitro findings are consistent with in vivo observations of known pathophysiological conditions associated with hLHR CAMs, but not hFSHR CAMs, and with promiscuous activation of hFSHR CAMs, but not hLHR CAMs. Computational experiments suggest that the mechanisms through which homologous mutations increase the basal activity of the hLHR and the hFSHR are similar. This is particularly true for the strongest CAMs like L460(3.43)R. Disparate properties of the hLHR versus hFSHR CAMs may, therefore, be due to differences in shape and electrostatics features of the solvent-exposed cytosolic receptor domains involved in the receptor-G protein interface rather than to differences in the nature of local perturbation at the mutation site or in the way local perturbation is transferred to the putative G protein binding domains.     

Thermal tolerance affects mutualist attendance in an ant–plant protection mutualism

The influence of native fauna on non-native plant population growth, size, and distribution is not well documented. Previous studies have shown that native insects associated with tall thistle (Cirsium altissimum) also feed on the leaves, stems, and flower heads of the Eurasian congener C. vulgare, thus limiting individual plant performance. In this study, we tested the effects of insect herbivores on the population growth rate of C. vulgare. We experimentally initiated invasions by adding seeds at four unoccupied grassland sites in eastern Nebraska, USA, and recorded plant establishment, survival, and reproduction. Cumulative foliage and floral herbivory reduced C. vulgare seedling density, and prevented almost any reproduction by C. vulgare in half the sites. The matrix model we constructed showed that this herbivory resulted in a reduction of the asymptotic population growth rate (λ), from an 88 % annual increase to a 54 % annual decline. These results provide strong support for the hypothesis that indigenous herbivores limit population invasion of this non-native plant species into otherwise suitable grassland habitat.     

Cytoskeleton disruption in J774 macrophages: Consequences for lipid droplet formation and cholesterol flux

Macrophages store excess unesterified cholesterol (free, FC) in the form of cholesteryl ester (CE) in cytoplasmic lipid droplets. The hydrolysis of droplet-CE in peripheral foam cells is critical to HDL-promoted reverse cholesterol transport because it represents the first step in cellular cholesterol clearance, as only FC is effluxed from cells to HDL. Cytoplasmic lipid droplets move within the cell utilizing the cytoskeletal network, but, little is known about the influence of the cytoskeleton on lipid droplet formation. To understand this role we employed cytochalasin D (cyt.D) to promote actin depolymerization in J774 macrophages. Incubating J774 with acetylated LDL creates foam cells having a 4-fold increase in cellular cholesterol content (30-40% cholesterol present as cholesteryl ester (CE)) in cytoplasmic droplets. Lipid droplets formed in the presence of cyt.D are smaller in diameter. CE-deposition and -hydrolysis are decreased when cells are cholesterol-enriched in the presence of cyt.D or latrunculin A, another cytoskeleton disrupting agent. However, when lipid droplets formed in the presence of cyt.D are isolated and incubated with an exogenous CE hydrolase, the CE is more rapidly metabolized compared to droplets from control cells. This is apparently due to the smaller size and altered lipid composition of the droplets formed in the presence of cyt.D. Cytoskeletal proteins found on CE droplets influence droplet lipid composition and maturation in model foam cells. In J774 macrophages, cytoskeletal proteins are apparently involved in facilitating the interaction of lipid droplets and a cytosolic neutral CE hydrolase and may play a role in foam cell formation. This article is part of a Special Issue entitled Advances in High Density Lipoprotein Formation and Metabolism: A Tribute to John F. Oram (1945-2010).     

Critical role of effector macrophages in mediating CD4-dependent alloimmune injury of transplanted liver parenchymal cells

Despite the recognition that humoral rejection is an important cause of allograft injury, the mechanism of Ab-mediated injury to allograft parenchyma is not well understood. We used a well-characterized murine hepatocellular allograft model to determine the mechanism of Ab-mediated destruction of transplanted liver parenchymal cells. In this model, allogeneic hepatocytes are transplanted into CD8-deficient hosts to focus on CD4-dependent, alloantibody-mediated rejection. Host serum alloantibody levels correlated with in vivo allospecific cytotoxic activity in CD8 knockout hepatocyte rejector mice. Host macrophage depletion, but not CD4(+) T cell, NK cell, neutrophil, or complement depletion, inhibited in vivo allocytotoxicity. Recipient macrophage deficiency delayed CD4-dependent hepatocyte rejection and inhibited in vivo allocytotoxicity without influencing alloantibody production. Furthermore, hepatocyte coincubation with alloantibody and macrophages resulted in Ab-dependent hepatocellular cytotoxicity in vitro. These studies are consistent with a paradigm of acute humoral rejection in which CD4(+) T cell-dependent alloantibody production results in the targeting of transplanted allogeneic parenchymal cells for macrophage-mediated cytotoxic immune damage. Consequently, strategies to eliminate recipient macrophages during CD4-dependent rejection pathway may prolong allograft survival.     

Testing the role of apoA-I,HDL, and cholesterol efflux in the atheroprotective action of low-level apoE expression

Low levels of transgenic mouse apolipoprotein E (apoE) suppress atherosclerosis in apoE knockout (apoE-/-) mice without normalizing plasma cholesterol. To test whether this is due to facilitation of cholesterol efflux from the vessel wall, we produced apoA-I-/-/apoE-/- mice with or without the transgene. Even without apoA-I and HDL, apoA-I-/-/apoE-/- mice had the same amount of aorta cholesteryl ester as apoE-/- mice. Low apoE in the apoA-I-/-/apoE-/- transgenic mice reduced aortic lesions by 70% versus their apoA-I-/-/apoE-/- siblings. To define the free cholesterol (FC) efflux capacity of lipoproteins from the various genotypes, sera were assayed on macrophages expressing ATP-binding cassette transporter A1 (ABCA1). Surprisingly, ABCA1 FC efflux was twice as high to sera from the apoA-I-/-/apoE-/- or apoE-/- mice compared with wild-type mice, and this activity correlated with serum apoA-IV. Immunodepletion of apoA-IV from apoA-I-/-/apoE-/- serum abolished ABCA1 FC efflux, indicating that apoAI-V serves as a potent acceptor for FC efflux via ABCA1. With increasing apoE expression, apoA-IV and FC acceptor capacity decreased, indicating a reciprocal relationship between plasma apoE and apoA-IV. Low plasma apoE (1-3 x 10(-8) M) suppresses atherosclerosis by as yet undefined mechanisms, not dependent on the presence of apoA-I or HDL or an increased capacity of serum acceptors for FC efflux.     

Blood lactate as a prognosticator of survival following hemorrhage in conscious swine

Arterial blood lactate concentration at the end of fixed volume hemorrhage was evaluated as a predictor of survival in unmedicated chronically instrumented immature swine. Compared to basal values, 8.4 +/- 4.5 mg/dl (means +/- SD, n = 52), hemorrhaged animals (n = 71) with a lactate of 43.9 +/- 37.1 mg/dl lived while animals (n = 65) with a lactate of 106.5 +/- 40.4 mg/dl died. Lactate concentration at the end of hemorrhage successfully predicted survival (81.0%). Prospective evaluation, using a blood lactate concentration of 125 mg/dl (n = 27), showed a predictive success of 81.0%. Arterial plasma lactate concentration at the end of hemorrhage thus may be used to predict survival in the conscious swine.     

Population genetics of grotto sculpin (Cottus specus), a new cave-adapted fish species

The grotto sculpin (Cottus specus) is a troglomorphic fish endemic to cave systems in Perry County, Missouri. These fish are state-threatened and were recently listed as federally endangered under the Endangered Species Act (ESA). Due to the unstable nature of the cave environment, grotto sculpins are highly susceptible to pollution via agricultural and waste runoff. Here we provide a summary of population genetic diversity within the range of the grotto sculpin. Samples from twenty-one cave, surface, and resurgence sites within and surrounding the Bois Brule drainage in Perry County, southeast Missouri, USA were collected and sequence data from the mitochondrial control were analyzed using analysis of molecular variance, maximum likelihood, parsimony, and Bayesian techniques. We found a substantial degree of divergence from surface populations of Midlands and Black River banded sculpin races (7.7–8.0 %). Within the Bois Brule drainage, two distinct lineages of grotto sculpin were identified which correspond with northern and southern cave system boundaries and may represent independent cave invasions.