The discovery of UK-369003, a novel PDE5 inhibitor with the potential for oral bioavailability and dose-proportional pharmacokinetics

This paper describes our recent efforts to design and synthesise potent and selective PDE5 inhibitors and the use of in vitro predictors of clearance, absorption and permeability to maximise the potential for dose-proportional pharmacokinetics and good oral bioavailability in man. Optimisation of the preclinical profile resulted in the identification of UK-369003 (19a) and its nomination as a clinical candidate. The clinical pharmacokinetic and safety profile has enabled us to progress the compound to test its efficacy in patients with lower urinary tract symptoms (LUTS) associated with benign prostatic hyperplasia (BPH) and a paper describing its efficacy has recently been published.     

Influence of seasonality on the genetic diversity of Vibrio parahaemolyticus in New Hampshire shellfish waters as determined by multilocus sequence analysis

Risk of gastric infection with Vibrio parahaemolyticus increases with favorable environmental conditions and population shifts that increase prevalence of infective strains. Genetic analysis of New Hampshire strains revealed a unique population with some isolates similar to outbreak-causing strains and high-level diversity that increased as waters warmed.     

Continuous biosynthesis of abscisic acid (ABA) may be required for maintaining dormancy of isolated embryos and intact seeds of Euonymus alatus

Euonymus alatus (Thunb.) Sieb. is a popular landscape plant in the United States due to its brilliant red fall foliage. It is also an important ornamental plant in many other areas of the world such as China, Japan and Europe. However, E. alatus is considered as a highly invasive plant species in the US. Mutation breeding can be used to create sterile, non-invasive cultivars. Seeds are the most commonly used explants for mutagen treatments, but E. alatus mature seeds possess prolonged dormancy and only a low percentage of them germinate even after 18?months of cold stratification. Here we report an immature embryo culture method for E. alatus ??Compactus?? to circumvent the seed dormancy problem. Also, we have found that activated charcoal, gibberellic acid (GA₃) and 6-benzyladenine (BA) can reduce the dormancy of isolated embryos, which suggests that abscisic acid (ABA) might play a role in controlling seed dormancy. We have further demonstrated that exogenous ABA enhances dormancy of isolated E. alatus embryos while fluridone, an inhibitor for ABA biosynthesis, can effectively break their dormancy. These results, particularly the effect of fluridone, suggest that continuous ABA biosynthesis plays an important role in controlling the dormancy of E. alatus seeds.     

Estrogen prevents cardiomyocyte apoptosis through inhibition of reactive oxygen species and differential regulation of p38 kinase isoforms

From human and animal studies, estrogen is known to protect the myocardium from an ischemic insult. However, there is limited knowledge regarding mechanisms by which estrogen directly protects cardiomyocytes. In this report, we employed an in vitro model, in which cultured rat cardiomyocytes underwent prolonged hypoxia followed by reoxygenation (H/R), to study the cardioprotective mechanism of estrogen. 17-beta-estradiol (E2) acting via estrogen receptors inhibited H/R-induced apoptosis of cardiomyocytes. Mitochondrial reactive oxygen species (ROS) generated from H/R activated p38alpha MAPK, and inhibition of p38alpha with SB203580 significantly prevented H/R-induced cell death. E2 suppressed ROS formation and p38alpha activation by H/R and concomitantly augmented the activity of p38beta. Unlike p38alpha, p38beta was little affected by H/R. Dominant negative p38beta protein expression decreased E2-mediated cardiomyocyte survival and ROS suppression during H/R stress. The prosurvival signaling molecule, phosphoinositol-3 kinase (PI3K), has previously been linked to cell survival following ischemia-reperfusion injury. Here, E2-activated PI3K was found to inhibit ROS generated from H/R injury, leading to inhibition of downstream p38alpha. We further linked these signaling pathways in that p38beta was activated by E2 stimulation of PI3K. Thus, E2 differentially modulated two major isoforms of p38, leading to cardiomyocyte survival. This was achieved by signaling through PI3K, integrating cell survival mediators.     

Genes within the Idd5 and Idd9/11 diabetes susceptibility loci affect the pathogenic activity of B cells in nonobese diabetic mice

Autoreactive T cells clearly mediate the pancreatic beta cell destruction causing type 1 diabetes (T1D). However, studies in NOD mice indicate that B cells also contribute to pathogenesis because their ablation by introduction of an Igmunull mutation elicits T1D resistance. T1D susceptibility is restored in NOD.Igmunull mice that are irradiated and reconstituted with syngeneic bone marrow plus NOD B cells, but not syngeneic bone marrow alone. Thus, we hypothesized some non-MHC T1D susceptibility (Idd) genes contribute to disease by allowing development of pathogenic B cells. Supporting this hypothesis was the finding that unlike those from NOD donors, engraftment with B cells from H2g7 MHC-matched, but T1D-resistant, nonobese-resistant (NOR) mice failed to restore full disease susceptibility in NOD.Igmunull recipients. T1D resistance in NOR mice is mainly encoded within the Idd13, Idd5.2, and Idd9/11 loci. B cells from NOD congenic stocks containing Idd9/11 or Idd5.1/5.2-resistance loci, respectively, derived from the NOR or C57BL/10 strains were characterized by suppressed diabetogenic activity. Immature autoreactive B cells in NOD mice have an impaired ability to be rendered anergic upon Ag engagement. Interestingly, both Idd5.1/5.2 and Idd9/11-resistance loci were found to normalize this B cell tolerogenic process, which may represent a mechanism contributing to the inhibition of T1D.     

Characterization of sequences and mechanisms through which ISE/ISS-3 regulates FGFR2 splicing

Alternative splicing of fibroblast growth factor receptor-2 (FGFR2) mutually exclusive exons IIIb and IIIc results in highly cell-type-specific expression of functionally distinct receptors, FGFR2-IIIb and FGFR2-IIIc. We previously identified an RNA cis-element, ISE/ISS-3, that enhanced exon IIIb splicing and silenced exon IIIc splicing. Here, we have performed comprehensive mutational analysis to define critical sequence motifs within this element that independently either enhance splicing of upstream exons or repress splicing of downstream exons. Such analysis included use of a novel fluorescence-based splicing reporter assay that allowed quantitative determination of relative functional activity of ISE/ISS-3 mutants using flow cytometric analysis of live cells. We determined that specific sequences within this element that mediate splicing enhancement also mediate splicing repression, depending on their position relative to a regulated exon. Thus, factors that bind the element are likely to be coordinately involved in mediating both aspects of splicing regulation. Exon IIIc silencing is dependent upon a suboptimal branchpoint sequence containing a guanine branchpoint nucleotide. Previous studies of exon IIIc splicing in HeLa nuclear extracts demonstrated that this guanine branchsite primarily impaired the second step of splicing suggesting that ISE/ISS-3 may block exon IIIc inclusion at this step. However, results presented here that include use of newly developed in vitro splicing assays of FGFR2 using extracts from a cell line expressing FGFR2-IIIb strongly suggest that cell-type-specific silencing of exon IIIc occurs at or prior to the first step of splicing.     

Naringenin is a novel inhibitor of Dictyostelium cell proliferation and cell migration

Naringenin is a flavanone compound that alters critical cellular processes such as cell multiplication, glucose uptake, and mitochondrial activity. In this study, we used the social amoeba, Dictyostelium discoideum, as a model system for examining the cellular processes and signaling pathways affected by naringenin. We found that naringenin inhibited Dictyostelium cell division in a dose-dependent manner (IC(50) approximately 20 microM). Assays of Dictyostelium chemotaxis and multicellular development revealed that naringenin possesses a previously unrecognized ability to suppress amoeboid cell motility. We also found that naringenin, which is known to inhibit phosphatidylinositol 3-kinase activity, had no apparent effect on phosphatidylinositol 3,4,5-trisphosphate synthesis in live Dictyostelium cells; suggesting that this compound suppresses cell growth and migration via alternative signaling pathways. In another context, the discoveries described here highlight the value of using the Dictyostelium model system for identifying and characterizing the mechanisms by which naringenin, and related compounds, exert their effects on eukaryotic cells.     

An ecological digest of the small hive beetle (Aethina tumida), a symbiont in honey bee colonies (Apis mellifera)

The small hive beetle (Aethina tumida Murray) is an endemic scavenger in colonies of western honey bee subspecies (Apis mellifera L.) inhabiting sub-Saharan Africa where it only occasionally damages host colonies. Such damage is usually restricted to weakened/diseased colonies or is associated with after absconding events (all bees, including the queen, leave the hive) due to behavioral resistance mechanisms of its host. In sharp contrast, the beetle has proven deleterious to honey bee colonies in introduced ranges of the United States and Australia. With this review we synthesize the existing data in a manner that allows us to discuss the beetle’s natural history from an ecological perspective. A thorough exploration of beetle ecology allows us to 1) illuminate the unique symbiotic relationship it and its host share and understand how this relationship is fostered, 2) place this relationship in context with those of other arthropods inhabiting social insect colonies, 3) understand its natural reliance on honey bee colonies, 4) predict its spread outside its native range, and 5) predict its effects on non-African honey bees and non-target species. Here we present an amalgamation of information that will contribute to a more thorough and appropriate understanding of not only small hive beetles as symbionts, but of social insect symbionts in general. Received 4 April 2005; revised 15 October 2005; accepted 18 October 2005.     

Detection of protein-protein interactions in the alkanesulfonate monooxygenase system from Escherichia coli

The two-component alkanesulfonate monooxygenase system utilizes reduced flavin as a substrate to catalyze a unique desulfonation reaction during times of sulfur starvation. The importance of protein-protein interactions in the mechanism of flavin transfer was analyzed in these studies. The results from affinity chromatography and cross-linking experiments support the formation of a stable complex between the flavin mononucleotide (FMN) reductase (SsuE) and monooxygenase (SsuD). Interactions between the two proteins do not lead to overall conformational changes in protein structure, as indicated by the results from circular dichroism spectroscopy in the far-UV region. However, subtle changes in the flavin environment of FMN-bound SsuE that occur in the presence of SsuD were identified by circular dichroism spectroscopy in the visible region. These data are supported by the results from fluorescent spectroscopy experiments, where a dissociation constant of 0.0022 +/- 0.0010 muM was obtained for the binding of SsuE to SsuD. Based on these studies, the stoichiometry for protein-protein interactions is proposed to involve a 1:1 monomeric association of SsuE with SsuD.     

Effect of decreased O2 supply on skeletal muscle oxygenation and O2 consumption during sepsis: role of heterogeneous capillary spacing and blood flow

One of the main aspects of the initial phase of the septic inflammatory response to a bacterial infection is abnormal microvascular perfusion, including decreased functional capillary density (FCD) and increased blood flow heterogeneity. On the other hand, one of the most important phenomena observed in the later stages of sepsis is an increased dependence of tissue O(2) utilization on the convective O(2) supply. This "pathological supply dependency" is associated with organ failure and poor clinical outcomes. Here, a detailed theoretical model of capillary-to-tissue O(2) transport during sepsis is used to examine the origins of abnormal supply dependency. With use of three-dimensional arrays of capillaries with heterogeneous spacing and blood flow, steady-state O(2) transport is simulated numerically during reductions in the O(2) supply. Increased supply dependency is shown to occur in sepsis for hypoxic (decreased hemoglobin O(2) saturation) and stagnant (decreased blood flow) hypoxia. For stagnant hypoxia, a reduction in FCD with decreasing blood flow is necessary to obtain the observed increase in supply dependency. Our results imply that supply dependency observed under normal conditions does not have its origin at the level of individual capillaries. In sepsis, however, diffusion limitation and shunting of O(2) by individual capillaries occur to a degree that is dependent on the heterogeneity of septic injury and the arrangement of capillary networks. Thus heterogeneous stoppage of individual capillaries is a likely factor in pathological supply dependency.