First dual M3 antagonists-PDE4 inhibitors: synthesis and SAR of 4,6-diaminopyrimidine derivatives

SAR around 4,6-diaminopyrimidine derivatives allowed the discovery of the first potent dual M(3) antagonists and PDE4 inhibitors. Various chemical modulations around that scaffold led to the discovery of ucb-101333-3 which is characterized by the most interesting profile on both targets.     

Insights into the architecture of the Ure2p yeast protein assemblies from helical twisted fibrils

The protein Ure2 from baker's yeast is associated with a heritable and transmissible phenotypic change in the yeast Saccharomyces cerevisiae. Such prion properties are thought to arise from the fact that Ure2p is able to self-assemble into insoluble fibrils. Assemblies of Ure2p are composed of full-length proteins in which the structure of the globular, functional, C-terminal domain is retained. We have carried out structural studies on full-length, wild-type Ure2p fibrils with a regularly twisted morphology. Using electron microscopy and cryo-electron microscopy with image analysis we show high-resolution images of the twisted filaments revealing details within the fibrillar structure. We examine these details in light of recent proposed models and discuss how this new information contributes to an understanding of the architecture of Ure2p yeast prion fibrils.     

Invasion of an asexual American water flea clone throughout Africa and rapid displacement of a native sibling species

The huge ecological and economic impact of biological invasions creates an urgent need for knowledge of traits that make invading species successful and factors helping indigenous populations to resist displacement by invading species or genotypes. High genetic diversity is generally considered to be advantageous in both processes. Combined with sex, it allows rapid evolution and adaptation to changing environments.We combined paleogenetic analysis with continent-wide survey of genetic diversity at nuclear and mitochondrial loci to reconstruct the invasion history of a single asexual American water flea clone (hybrid Daphnia pulexxDaphnia pulicaria) in Africa. Within 60 years of the original introduction of this invader, it displaced the genetically diverse, sexual population of native D. pulex in Lake Naivasha (Kenya), despite a formidable numerical advantage of the local population and continuous replenishment from a large dormant egg bank. Currently, the invading clone has spread throughout the range of native African D. pulex, where it appears to be the only occurring genotype.The absence of genetic variation did not hamper either the continent-wide establishment of this exotic lineage or the effective displacement of an indigenous and genetically diverse sibling species.     

Regulation of the intracellular distribution, cell surface expression, and protein levels of AMPA receptor GluR2 subunits by the monocarboxylate transporter MCT2 in neuronal cells

The neuronal monocarboxylate transporter, MCT2, is not only an energy substrate carrier but it is also purported to be a binding partner for the α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptor GluR2 subunit. To unravel a putative role of MCT2 in the regulation of GluR2 subcellular distribution, Neuro2A cells and primary cultures of mouse cortical neurons were co-transfected with plasmids containing sequences to express the fluorescent proteins mStrawberry (mStb)-fused MCT2 and Venus-fused GluR2. Subsequently, their subcellular distribution was visualized by fluorescence microscopy. GluR2 was led to form perinuclear and dendritic clusters together with MCT2 when co-transfected in Neuro2A cells or in neurons, following the original distribution of MCT2. MCT2 co-transfection had no effect on the intracellular distribution of several other post-synaptic proteins, although it partially affected the intracellular distribution of GluR1 similarly to GluR2. Both cell surface and total protein expression levels of GluR2 were significantly reduced by co-expression with MCT2. Finally, partial perinuclear and dendritic co-localization between MCT2 and Rab8, a member of the small GTPase family involved in membrane trafficking of AMPA receptors, was also observed in co-transfected neurons. These results suggest that MCT2 could influence AMPA receptor trafficking within neurons by modulating GluR2 sorting between different subcellular compartments.     

Genotype-dependent interactions among sympatric Microcystis strains mediated by Daphnia grazing

Natural populations of the bloom forming cyanobacterium Microcystis are typically composed of several distinct genotypes. Using Microcystis strains that differ in growth rate, microcystin production and colony formation, we conducted a laboratory experiment in the presence and absence of a grazer, the water flea Daphnia, to investigate whether interactions among strains can be predicted from functional traits, and whether the outcome of competition between strains is influenced by a grazer. Two toxic and two non‐toxic Microcystis strains, isolated from a single lake, were grown during four weeks as single strains, in all possible combinations of two strains and all together, in the presence and absence of Daphnia magna. The relative abundance of strains in the populations was assessed using denaturing gradient gel electrophoresis, and the growth rate of each strain in mixed populations was compared to its growth rate in monoculture to determine interactions between strains. The observed interactions were strain‐specific, and the relative abundances of strains in mixed populations could be partially explained by taking toxicity and colony formation into account. Importantly, some of the interactions were strongly altered by the presence of Daphnia. Daphnia induced colony formation in one strain, which then became a better competitor. Daphnia grazing also caused a higher evenness in the populations, both through a weakening of competitive interactions as well as by facilitation effects. Strong facilitation effects were due to non‐toxic strains benefiting from the protection offered by toxic strains in the presence of predation. Overall, our results emphasize the presence of strong competitive interactions between Microcystis strains in the absence of grazing, whereas indirect positive interactions are prevalent in the presence of a generalist grazer. Our results suggest that differences in functional traits and grazer‐mediated facilitation effects may enhance coexistence of Microcystis strains, including toxic and non‐toxic strains.     

Oxidative burst inhibitory and cytotoxic amides and lignans from the stem bark of Fagara heitzii (Rutaceae)

Two amides, heitziamide A and heitziamide B and two phenylethanoids, heitziethanoid A and heitziethanoid B together with thirteen known compounds were isolated from F. heitzii (Letouzey). The structures of all compounds were established by spectroscopic analysis.Nine compounds were evaluated for oxidative burst inhibitory activity in a chemoluminescence assay and for cytotoxicity against PC-3 prostate cancer cells.All compounds exhibited a clear suppressive effect on phagocytosis response upon activation with serum opsonized zymosan at the range of IC₅₀ = 2.0–6.5 μM, but no cytotoxic effect was observed (IC₅₀ > 100 μM).     

Polyphenols isolated from antiradical extracts of Mallotus metcalfianus

Six flavonoids including two new flavones, luteolin 7-O-(4″-O-(E)-coumaroyl)-β-glucopyranoside), chrysoeriol-7-O-(4″-O-(E)-coumaroyl)-β-glucopyranoside) and a mixture of two pairs of diastereoisomeric flavonolignans, (±)-hydnocarpin 7-O-(4″-O-(E)-coumaroyl)-β-glucopyranoside)/(±)-hydnocarpin-D 7-O-(4″-O-(E)-coumaroyl)-β-glucopyranoside) with a 2:1 ratio were isolated from the whole plant of Mallotus metcalfianus Croizat, in addition to 10 known compounds. Their structures were evaluated on the basis of different spectroscopic methods, including extensive 1D and 2D NMR spectroscopy. Some extracts have moderate antimicrobial properties and interesting antiradical (DPPH) activity, as well as some compounds isolated from this species. Tannins were also identified in some active extracts.     

Peculiar inhibition of human mitochondrial aspartyl-tRNA synthetaseby adenylate analogs

Human mitochondrial aminoacyl-tRNA synthetases (mt-aaRSs), the enzymes which esterify tRNAs with the cognate specific amino acid, form mainly a different set of proteins than those involved in the cytosolic translation machinery. Many of the mt-aaRSs are of bacterial-type in regard of sequence and modular structural organization. However, the few enzymes investigated so far do have peculiar biochemical and enzymological properties such as decreased solubility, decreased specific activity and enlarged spectra of substrate tRNAs (of same specificity but from various organisms and kingdoms), as compared to bacterial aaRSs. Here the sensitivity of human mitochondrial aspartyl-tRNA synthetase (AspRS) to small substrate analogs (non-hydrolysable adenylates) known as inhibitors of Escherichia coli and Pseudomonas aeruginosa AspRSs is evaluated and compared to the sensitivity of eukaryal cytosolic human and bovine AspRSs. l-aspartol-adenylate (aspartol-AMP) is a competitive inhibitor of aspartylation by mitochondrial as well as cytosolic mammalian AspRSs, with K_i values in the micromolar range (4–27 μM for human mt- and mammalian cyt-AspRSs). 5′-O-[N-(l-aspartyl)sulfamoyl]adenosine (Asp-AMS) is a 500-fold stronger competitive inhibitor of the mitochondrial enzyme than aspartol-AMP (10 nM) and a 35-fold lower competitor of human and bovine cyt-AspRSs (300 nM). The higher sensitivity of human mt-AspRS for both inhibitors as compared to either bacterial or mammalian cytosolic enzymes, is not correlated with clear-cut structural features in the catalytic site as deduced from docking experiments, but may result from dynamic events. In the scope of new antibacterial strategies directed against aaRSs, possible side effects of such drugs on the mitochondrial human aaRSs should thus be considered.     

Relationship of cryptorchidism with sex ratios and litter sizes in 12 dog breeds

The aim of this study was to identify the influence of genetic carriership for cryptorchidism on litter sizes and sex ratios in the offspring. Weaning data of 11,230 litters in 12 purebred dog breeds were evaluated. Parents were classified as cryptorchidism 'carriers' (C) when at least one of their offspring was found cryptorchid. Subsequently the effects of 'carrier' and 'non-carrier' (NC) parents on their litters were studied. In litters from C x C parents we found an increased number of males per litter in all breeds, a reduced number of females per litter in 8 breeds and an increased litter size in 11 breeds in comparison with litters from NC x NC parents. Over all breeds the effects on litter size, on number of males per litter and on sex ratio were highly significant. Mixed litters from C x NC and NC x C did not show these effects and were not significantly different from the NC x NC offspring. Our results suggest a general mechanism in the dog species which causes cryptorchidism as well as increased male/female ratios and increased litter sizes. A consequence of such a mechanism is that selection in favor of increasing reproduction output frustrates selective efforts to eliminate cryptorchidism.     

Hierarchy of factors affecting behavioural signs used for oestrus detection of Holstein and Normande dairy cows in a seasonal calving system

As oestrous expression of dairy cows has decreased over the last decades oestrus detection has become more difficult. The objective of this study is to identify the main factors that affect oestrus detection in seasonal calving dairy cows, and to establish their relative importance. In each of 5 years 36 Normande and 36 Holstein cows were assigned to a Low or High winter-feeding level group. Half of each group was then assigned to a Low or High pasture-feeding group. The Low-Low strategy resulted in the lowest milk yield and the greatest body condition (BC) loss from calving to nadir BC score (6302 kg; -0.98 unit). The High-High strategy had the converse effect (7549 kg; -0.75 units). Low-High and High-Low strategies had intermediate values. The Normande cows had lower milk yield and BC loss than Holstein cows (6153 kg versus 7620 kg; -0.82 unit versus -1.20 unit). A database of 415 observed spontaneous oestruses was created. Oestruses were classified according to detection signs: (1) standing to be mounted, (2) mounting without standing, (3) other signs without standing or mounting (slight signs). Presence of another cow in oestrus, access to pasture, Normande breed and Low-Low strategy increased standing detection. In the Normande breed, 97% of oestruses were detected by standing while combining the presence of a herdmate in oestrus and access to pasture with a milk production of less than 6550 kg. Holstein cows had a higher frequency of slight signs oestruses than Normande ones, which was associated with a decreased subsequent calving rate (P<0.05). In multiparous Holstein cows, the odds of slight signs detection was multiplied by 7.8 for the High-High group in comparison with the Low-Low group (P<0.05). In our study milk yield had an effect on oestrus detection which was not explained by BC loss. As High-High cows produced more milk than others, we logically found that an increase in milk yield increased slight signs detection. Conversely, as they lost less BC than others, BC loss improved the chance of standing or mounting detection. These two results show that an increase in milk yield may reduce oestrous behaviour even if BC loss is moderate. Oestrus detection is crucial in seasonal compact calving systems. High phenotypic milk yields appear unsuitable with such systems in regard to depressed oestrous behaviour.