Identification of a selective nonpeptide antagonist of the anaphylatoxin C3a receptor that demonstrates antiinflammatory activity in animal models

The anaphylatoxin C3a is a potent chemotactic peptide and inflammatory mediator released during complement activation which binds to and activates a G-protein-coupled receptor. Molecular cloning of the C3aR has facilitated studies to identify nonpeptide antagonists of the C3aR. A chemical lead that selectively inhibited the C3aR in a high throughput screen was identified and chemically optimized. The resulting antagonist, N(2)-[(2,2-diphenylethoxy)acetyl]-L-arginine (SB 290157), functioned as a competitive antagonist of (125)I-C3a radioligand binding to rat basophilic leukemia (RBL)-2H3 cells expressing the human C3aR (RBL-C3aR), with an IC(50) of 200 nM. SB 290157 was a functional antagonist, blocking C3a-induced C3aR internalization in a concentration-dependent manner and C3a-induced Ca(2+) mobilization in RBL-C3aR cells and human neutrophils with IC(50)s of 27.7 and 28 nM, respectively. SB 290157 was selective for the C3aR in that it did not antagonize the C5aR or six other chemotactic G protein-coupled receptors. Functional antagonism was not solely limited to the human C3aR; SB 290157 also inhibited C3a-induced Ca(2+) mobilization of RBL-2H3 cells expressing the mouse and guinea pig C3aRS: It potently inhibited C3a-mediated ATP release from guinea pig platelets and inhibited C3a-induced potentiation of the contractile response to field stimulation of perfused rat caudal artery. Furthermore, in animal models, SB 290157, inhibited neutrophil recruitment in a guinea pig LPS-induced airway neutrophilia model and decreased paw edema in a rat adjuvant-induced arthritis model. This selective antagonist may be useful to define the physiological and pathophysiological roles of the C3aR.     

Elevated CO22 and Leaf Longevity in the C44 Grassland-Dominant Andropogon gerardii

In central U.S. grasslands, plant and ecosystem responses to elevated CO2 are most pronounced when water availability is limited. In a northeast Kansas grassland, responses to elevated CO2 in leaf area, number, development, and longevity were quantified for the tallgrass prairie dominant, Andropogon gerardii. Plants were grown in open-top chambers (OTCs) modified to limit water availability and to maximize responses to elevated CO2. In OTCs with elevated (x2 ambient) levels of CO2, aboveground biomass production and leaf water potentials were increased significantly compared with those of plants in OTCs with ambient CO2. There were no differences in leaf area or leaf number per tiller in A. gerardii in elevated compared with ambient OTCs. However, leaf area in adjacent unchambered plots with greater water availability was significantly higher than in the OTCs. The time required for developing leaves to achieve maximum leaf area was reduced by 29%, and the period of time until leaves senesced was increased by 20% for plants exposed to elevated compared with ambient CO2. Thus, leaves of this C4 grass species expanded more rapidly (6 d) and remained green longer (9 d) when exposed to elevated CO2. Such CO2-mediated increases in leaf longevity in the dominant species may allow this grassland to respond more opportunistically to temporally variable rainfall patterns in high-CO2 environments. These responses should be included in leaf-based simulation models that attempt to mechanistically link physiological alterations to predicted canopy responses to increased CO2.     

Identification of major proteins in maize egg cells

In most flowering plants, the female gametophyte develops in an ovule deeply embedded in the ovary. Through double fertilization, the egg cell fuses with the sperm cell, resulting in a zygote, which develops into the embryo. In the present study, we analyzed egg cell lysates by polyacrylamide gel electrophoresis and subsequent mass spectrometry-based proteomics technology, and identified major protein components expressed in the egg cell. The identified proteins included three cytosolic enzymes of the glycolytic pathway, glyceraldehyde-3-phosphate dehydrogenase, 3-phosphoglycerate kinase and triosephosphate isomerase, two mitochondrial proteins, the ATP synthase beta-subunit and an adenine nucleotide transporter, and annexin p35. In addition, expression levels of these proteins in the egg cell were compared with those in the early embryo, the central cell and the suspension cell. Annexin p35 was highly expressed only in the egg cell, and glyceraldehyde-3-phosphate dehydrogenase, 3-phosphoglycerate kinase and the adenine nucleotide transporter were expressed at higher levels in egg cells than in central and cultured cells. These results indicate that annexin p35 in the egg cell and zygote is involved in the exocytosis of cell wall materials, which is induced by a fertilization-triggered increase in cytosolic Ca2+ levels, and that the egg cell is rich in an enzyme subset for the energy metabolism.     

NAB2, a corepressor of EGR-1, inhibits vascular endothelial growth factor-mediated gene induction and angiogenic responses of endothelial cells

In this study we have investigated the role of a specific corepressor of EGR-1, NAB2, to down-regulate vascular endothelial growth factor (VEGF)-induced gene expression in endothelial cells and to inhibit angiogenesis. Firstly, we show a reciprocal regulation of EGR-1 and NAB2 following VEGF treatment. During the initial phase EGR-1 is rapidly induced and NAB2 levels are down-regulated. This is followed by a reduction of EGR-1 and a concomitant increase of NAB2. Secondly, using the tissue factor gene as a readout for VEGF-induced and EGR-1-regulated gene expression we demonstrate that NAB2 can completely block VEGF-induced tissue factor reporter gene activity. Thirdly, by adenovirus-mediated expression we show that NAB2 inhibits up-regulation of tissue factor, VEGF receptor-1, and urokinase plasminogen activator mRNAs even when a combination of VEGF and bFGF is used for induction. In addition, NAB2 overexpression significantly reduced tubule and sprout formation in two different in vitro angiogenesis assays and largely prevented the invasion of cells and formation of vessel-like structures in the murine Matrigel model. These data suggest that NAB2 regulation represents a mechanism to guarantee transient EGR-1 activity following exposure of endothelial cells to VEGF and that NAB2 overexpression could be used to inhibit signals involved in the early phase of angiogenesis.     

Detection of the embryotoxic potential of cyclophosphamide by using a combined system of metabolic competent cells and embryonic stem cells

In order to develop a method for detecting metabolism-mediated embryotoxicity, differentiating embryonic stem (ES) cells were exposed to the well-known proteratogen, cyclophosphamide (CPA). CPA was tested in a scientifically validated embryonic stem-cell test (EST), and in the newly developed reporter-gene assay for developmental cardiotoxicity. Both assays gave false-negative results. Because no metabolic competence (cytochrome P450 [CYP] activity) was found in the ES cells under the selected culture conditions, a simple biotransformation system was combined with the reporter-gene assay. As the metabolic pathway of CPA is well characterised, the genetically engineered mammalian cell line V79, transfected with CYP2B1 cDNA, was selected as a biotransformation system. CYP2B1 is responsible for transforming CPA into teratogenically active metabolites. The supernatants of genetically engineered V79 cells were analysed in the reporter-gene assay for developmental cardiotoxicity. In preliminary experiments, the combined system was able to detect the embryotoxic potential of the proteratogen, CPA.     

Phenylbutyrates as potent,orally bioavailable vitronectin receptor (integrin alphavbeta3) antagonists

In our continuing efforts to identify small molecule vitronectin receptor antagonists, we have discovered a series of phenylbutyrate derivatives, exemplified by 16, which have good potency and excellent oral bioavailability (approximately 100% in rats). This new series is derived conceptually from opening of the seven-membered ring of SB-265123.     

Can one unrestricted meal buffer the effects of previous pre-meal intervals on the feeding behaviour of sheep?

Varying the time since the last meal is one means of manipulating feeding motivation. In order to use this method effectively it is necessary to know whether and the extent to which effects of one pre-meal interval are carried over to affect the behaviour during the following meals. Pre-meal interval (PMI) is defined here for practical purposes, for short meals, as the time between the start of two successive meals. The possibility that one unrestricted meal might buffer the effects of an 8h as opposed to a 4h PMI on aspects of feeding behaviour was studied with eight Scottish Blackface sheep. They were fed on a regime in which they were given access to food until they finished their meal and lay down (this always occurred within 60min) at which time the remaining food was withdrawn. Feeding behaviour was recorded during the meal after these 4 and 8h intervals, as well as during the following meal 4h later.At a meal after a PMI of 8h, compared to 4h, sheep had a higher intake per meal (mean+/-S.T.D. for 8 and 4h PMI, respectively: 604.4+/-78.8 and 430.1+/-100.9g; P<0.001), a longer meal duration (27.1+/-7.5 and 21.8+/-8.1min; P<0.001), and a tendency for a higher intake rate (23.8+/-6.2 and 21.9+/-8.2g/min; P=0.11). During the following meal 4h later these differences were smaller, but intake per meal still tended to be higher (430.8+/-81.5 and 338.5+/-45.6g; P<0.06) for sheep who had previously had the 8h PMI. Meal duration (21.9+/-7.2 and 20.6+/-7.08min; P=0.28) and intake rate (21.2+/-6.1 and 18.7+/-7.2g/min; P=0.13) were no longer different.A single meal after the different PMIs reduced differences in all three aspects of feeding behaviour observed during the subsequent meal, 4h later, but differences in intake per meal were still apparent. It is suggested that an additional meal may overcome the carry-over effect.     

Combined phylogenetic analysis in the Rubiaceae-Ixoroideae: morphology, nuclear and chloroplast DNA data

Parsimony analyses of morphology, restriction sites of the cpDNA, sequences from the nuclear, ribosomal internal transcribed spacer (ITS), and the chloroplast gene rbcL were performed to asses tribal and generic relationships in the subfamily Ixoroideae (Rubiaceae). The tribes Vanguerieae and Alberteae (Antirheoideae) are clearly part of Ixoroideae, as are some Cinchonoideae taxa. Pavetteae should exclude Ixora and allies, which should be recognized as the tribe Ixoreae. Heinsenia, representing Aulacocalyceae, is part of Gardenieae, as is Duperrea, a genus earlier placed in Pavetteae. Posoqueria and Bertiera and the taxa in the subtribe Diplosporinae should be excluded from Gardenieae. Bertiera and three Diplosporinae taxa are part of Coffeeae, while Cremaspora (Diplosporinae) is best housed in a tribe of its own, Cremasporeae. The mangrove genus Scyphiphora, recently placed in Diplosporinae, is closer to Ixoreae and tentatively included there. The combined analysis resulted in higher resolution compared to the separate analyses, exemplifying that combined analyses can remedy the incapability of one data set to resolve portions of a phylogeny. Twenty-four new rbcL sequences representing all five Ixoroideae tribes (sensu Robbrecht) are presented.     

Estimating divergence times in large phylogenetic trees

A new method, PATHd8, for estimating ultrametric trees from trees with edge (branch) lengths proportional to the number of substitutions is proposed. The method allows for an arbitrary number of reference nodes for time calibration, each defined either as absolute age, minimum age, or maximum age, and the tree need not be fully resolved. The method is based on estimating node ages by mean path lengths from the node to the leaves but correcting for deviations from a molecular clock suggested by reference nodes. As opposed to most existing methods allowing substitution rate variation, the new method smoothes substitution rates locally, rather than simultaneously over the whole tree, thus allowing for analysis of very large trees. The performance of PATHd8 is compared with other frequently used methods for estimating divergence times. In analyses of three separate data sets, PATHd8 gives similar divergence times to other methods, the largest difference being between crown group ages, where unconstrained nodes get younger ages when analyzed with PATHd8. Overall, chronograms obtained from other methods appear smoother, whereas PATHd8 preserves more of the heterogeneity seen in the original edge lengths. Divergence times are most evenly spread over the chronograms obtained from the Bayesian implementation and the clock-based Langley-Fitch method, and these two methods produce very similar ages for most nodes. Evaluations of PATHd8 using simulated data suggest that PATHd8 is slightly less precise compared with penalized likelihood, but it gives more sensible answers for extreme data sets. A clear advantage with PATHd8 is that it is more or less instantaneous even with trees having several thousand leaves, whereas other programs often run into problems when analyzing trees with hundreds of leaves. PATHd8 is implemented in freely available software.