Novel triazines as potent and selective phosphodiesterase 10A inhibitors

The identification of highly potent and orally active triazines for the inhibition of PDE10A is reported. The new analogs exhibit low-nanomolar potency for PDE10A, demonstrate high selectivity against all other members of the PDE family, and show desired drug-like properties. Employing structure-based drug design approaches, we investigated the selectivity of PDE10A inhibitors against other known PDE isoforms, by methodically exploring the various sub-regions of the PDE10A ligand binding pocket. A systematic assessment of the ADME and pharmacokinetic properties of the newly synthesized compounds has led to the design of drug-like candidates with good brain permeability and desirable drug kinetics (t(1/2), bioavailability, clearance). Compound 66 was highly potent for PDE10A (IC(50)=1.4nM), demonstrated high selectivity (>200×) for the other PDEs, and was efficacious in animal models of psychoses; reversal of MK-801 induced hyperactivity (MED=0.1mg/kg) and conditioned avoidance responding (CAR; ID(50)=0.2mg/kg).     

Aggregative response in bats: prey abundance versus habitat

In habitats where prey is either rare or difficult to predict spatiotemporally, such as open habitats, predators must be adapted to react effectively to variations in prey abundance. Open-habitat foraging bats have a wing morphology adapted for covering long distances, possibly use information transfer to locate patches of high prey abundance, and would therefore be expected to show an aggregative response at these patches. Here, we examined the effects of prey abundance on foraging activities of open-habitat foragers in comparison to that of edge-habitat foragers and closed-habitat foragers. Bat activity was estimated by counting foraging calls recorded with bat call recorders (38,371 calls). Prey abundance was estimated concurrently at each site using light and pitfall traps. The habitat was characterized by terrestrial laser scanning. Prey abundance increased with vegetation density. As expected, recordings of open-habitat foragers clearly decreased with increasing vegetation density. The foraging activity of edge- and closed-habitat foragers was not significantly affected by the vegetation density, i.e., these guilds were able to forage from open habitats to habitats with dense vegetation. Only open-habitat foragers displayed a significant and proportional aggregative response to increasing prey abundance. Our results suggest that adaptations for effective and low-cost foraging constrains habitat use and excludes the guild of open-habitat foragers from foraging in habitats with high prey abundance, such as dense forest stands.     

Elevated air carbon dioxide concentrations increase dissolved carbon leaching from a cropland soil

Terrestrial sources of nitrogen (N), particularly N-fixing alder, may be important for sustaining production in headwater streams that typically lack substantial subsidies of marine-derived nutrients from spawning salmon yet support upstream-dispersing juvenile salmonids. However, other physiographic characteristics, such as watershed slope and topographic wetness, also control transport of nutrients to streams and may confound apparent linkages between alder and stream N. Seasonal patterns in precipitation and temperature may interact with watershed characteristics to modulate stream N availability. We empirically modeled the effect of alder cover and other watershed physiographic variables on stream N and contrasted these relationships over the growing season among 25 first-order streams from the lower Kenai Peninsula, Alaska. For each date, percent alder cover, mean topographic wetness, and mean slope were used as watershed predictors of NO _x –N concentration (nitrate?+?nitrite) and daily NO _x –N yield using Generalized Additive Models (GAM) and compared using Akaike’s Information Criterion (AIC_c). Alder cover was the only probable model and explained 75–96% of the variation in NO _x –N concentration and 83–89% of the variation in daily NO _x –N yield. The relationship between alder and both NO _x –N concentration and daily NO _x –N yield changed from constant inputs in May across the range of alder cover (linear fit) to increasing inputs in July and September (non-linear fits) implying that high-alder watersheds were N-saturated. The strong linkage between alder and stream N coupled with the concurrent timing of maximum stream N from alder in the spring to salmon fry emergence indicates the potential importance of this subsidy to headwater stream ecosystems.     

alpha2,3/alpha2,6-Sialylation of N-glycans: non-synonymous signals with marked developmental regulation in bovine reproductive tracts

The glycan part endows cellular glycoconjugates with significant potential for biological recognition. N-Glycan branches often end with alpha2,3/alpha2,6-sialylation, posing the question whether and how placement of the sialic acid at 3 - or 6 -acceptor positions of galactose has cell biological relevance. As attractive model to study developmental regulation we monitored the expression of alpha2,3/alpha2,6-sialylated determinants in fetal and adult bovine testes and ovaries by lectin histochemistry. Distinct expression patterns were detected in both organ types. Oocyte staining, as a prominent example, was restricted to the presence of alpha2,6-sialylated glycans. Treatment with sialidase abolished binding and thus excluded sulfate esters as lectin targets. We added computer simulations to rationalize the observed evidence for non-random expression of the two closely related sialylgalactose isomers. Extensive molecular mechanics and molecular dynamics calculations reveal that the seemingly minor shift of the glycosidic bond from the alpha2,3 position to the alpha2,6 configuration causes significant shape and flexibility changes. They give each disaccharide its own characteristic meaning as signal in the sugar code.     

Tumor-associated antigen 90K/Mac-2-binding protein: possible role in colon cancer

The tumor-associated antigen 90K (TAA90K)/Mac-2-binding protein implicated in cancer progression and metastasis is modified by beta1-6 branched N-linked oligosaccharides in colon cancer cells, glycans shown to contribute to cancer metastasis. To elucidate the role of TAA90K in colon cancer, we examined its expression and function in human colon tumors and colon carcinoma cell lines. Immunohistochemical analyses of colon tumors revealed elevated expression of TAA90K in all samples analyzed compared to normal colon. To examine the function of TAA90K in colon cancer, we carried out protein and cell binding assays using TAA90K-His purified from HT-29 cells colon carcinoma cells infected with recombinant vaccinia virus expressing TAA90K containing a C-terminal poly-histidine tag. TAA90K-His bound to fibronectin, collagen IV, laminins-1, -5, and -10 and galectin-3 (Mac-2) but poorly to collagen I and galectin-1. As expected, binding of TAA90K to galectin-3 was dependent on carbohydrate since it was inhibitable by lactose and asiolofetuin, and a TAA90K-His glycoform purified from HT-29 cells treated with the glycosylation inhibitor 1-deoxymannojirimycin bound poorly to galectin-3. Unlike TAA90K isolated from other cell types, TAA90K-His isolated from colon cancer cells failed to mediate adhesion of colon cancer and normal cell lines, possibly due to cell-type specific glycosylation of TAA90K-His and/or its putative cellular receptor. However, at low concentrations, TAA90K-His enhanced galectin-3-mediated HT-29 cell adhesion while at high concentrations, it inhibited cell adhesion. Thus, a possible mechanism by which TAA90K may contribute to colon cancer progression is by modulating tumor cell adhesion to extracellular proteins, including galectin-3.     

DNA uracil repair initiated by the archaeal ExoIII homologue Mth212 via direct strand incision

No genes for any of the known uracil DNA glycosylases of the UDG superfamily are present in the genome of Methanothermobacter thermautotrophicus ΔH, making it difficult to imagine how DNA-U repair might be initiated in this organism. Recently, Mth212, the ExoIII homologue of M. thermautotrophicus ΔH has been characterized as a DNA uridine endonuclease, which suggested the possibility of a novel endonucleolytic entry mechanism for DNA uracil repair. With no system of genetic experimentation available, the problem was approached biochemically. Assays of DNA uracil repair in vitro, promoted by crude cellular extracts, provide unequivocal confirmation that this mechanism does indeed operate in M. thermautotrophicus ΔH.     

N-domain of human adhesion/growth-regulatory galectin-9: Preference for distinct conformers and non-sialylated N-glycans and detection of ligand-induced structural changes in crystal and solution

Human tandem-repeat-type galectin-9 is a potent adhesion/growth-regulatory effector via lectin capacity of its N- and C-terminal domains. This bioactivity prompted further crystallographic study of the N-domain, combined with analysis in solution. Binding of lactose markedly increased the N-domain's resistance to thermal denaturation. Crystallography revealed its intimate contact profile, besides detecting an extension of the β-sandwich fold by an antiparallel β-strand F0 aligned to the C-terminal F1 strand. Ligand accommodation in its low-energy conformation leads to a movement of Arg87's side chain. As consequence, the ligand's glucose moiety and Arg87 become hydrogen bonded. The resulting predictions for spatial parameters in solution were verified by determining (a) the pattern of magnetization transfer from the protein to protons of lactose and Forssman disaccharide by NMR spectroscopy and (b) the ellipticity changes at wavelengths characteristic for Trp/Tyr residues in near-UV CD spectroscopy. Whereas solid-phase assays confirmed a previously noted tendency for homo- and heterotypic aggregation, gel filtration and ultracentrifugation disclosed monomeric status in solution, in line with crystallographic data. Using cell mutants with defects in glycosylation, this lectin domain was shown to preferentially bind N-glycans without α2,3-sialylation. Since proximal promoter sequences were delineated to diverge markedly among galectin genes and resulting differences in expression profiles were exemplarily documented immunohistochemically, the intrafamily diversification appears to have assigned this protein to a characteristic expression and activity profile among galectins. Our data thus take the crystallographic information to the level of the lectin in solution and in tissues by a strategic combination of spectroscopic and cell/histochemical assays.     

Effects of Forage Quality and Cell Wall Constituents of Bermuda Grass on Biochemical Conversion to Ethanol

Bermuda grass is an attractive candidate as a feedstock for biofuel production because over four million hectares of Bermuda grass are already grown for forage in the Southern USA. Because both rumen digestion and biochemical conversion to ethanol depend upon enzymatic conversion of the cell wall polysaccharides into fermentable sugars, it is probable that grasses bred for increased forage quality would be more amenable for ethanol production. However, it is not known how variation in rumen digestibility and cell wall/fiber components correlates with efficiency of conversion to ethanol via fermentation. The objective of this research was to determine relationships between ethanol production evaluated by simultaneous saccharification and fermentation (SSF), 72-h in vitro ruminal dry matter digestibility (IVDMD), in vitro ruminal gas production after 24 and 96 h, and biomass composition for 50 genetically diverse Bermuda grass accessions. The Bermuda grass samples were subjected to standard 72-h IVDMD and forage fiber analyses. Also, in separate labs, gas production was measured in sealed volume-calibrated vials after 24 (NNG24) and 96 h (NNG96) of in vitro fermentation by ruminal fluid; ethanol and pentose sugar productions were measured from a bench-top SSF procedure; cell wall constituents were determined by the Uppsala Dietary Fiber Method; and total nitrogen, carbon, and ash concentrations were determined by using the LECO combustion method. Ethanol production was moderately correlated with IVDMD (r?=?0.55) and NNG96 (r?=?0.63) but highly correlated with NNG24 (r?=?0.93). Ethanol was negatively correlated with neutral detergent fiber (NDF; r?=??0.53) and pentose sugars (r?=??0.60), but not correlated with glucose content. Regression models indicated that NDF and cell wall pentose sugar concentrations had significant negative effects on ethanol production. Variation among entries for IVDMD was affected by variability of NDF, pentose sugar concentrations, and biomass nitrogen content. Variation in Klason lignin content had only minor negative impacts on ethanol production and IVDMD. Biochemical conversion efficiency of Bermuda grass by SSF can be best estimated by NNG24 but not by IVDMD.