Conformationally constrained NR2B selective NMDA receptor antagonists derived from ifenprodil: Synthesis and biological evaluation of tetrahydro-3-benzazepine-1,7-diols

NR2B selective NMDA receptor antagonists with tetrahydro-3-benzazepine-1,7-diol scaffold have been designed by formal cleavage and reconstitution of the piperidine ring of the lead compound ifenprodil (1). The secondary amine 10 represents the central building block for the synthesis of more than 25 tetrahydro-3-benzazepin-1-ols. Generally 7-hydroxy derivatives display higher NR2B receptor affinities than the corresponding 7-benzyloxy compounds. A distance of four atoms (five bond lengths) between the basic amino group and the terminal aryl moiety led to highest NR2B affinity. 3-(4-Phenylbutyl)-2,3,4,5-tetrahydro-1H-3-benzazepine-1,7-diol (WMS-1410, 25) represents the most promising NR2B antagonist of this series showing a K(i)-value of 14nM. Compound 25 reveals excellent selectivity over more than 100 further relevant target proteins, antagonizes glutamate induced excitotoxicity (IC(50)=18.4nM) and is metabolically more stable than ifenprodil. Up to a dose of 100mg/kg 25 is well tolerated by mice and it shows dose dependent analgesic activity in the late neuropathic pain phase of the formalin assay.     

Loss of the p53/p63 regulated desmosomal protein Perp promotes tumorigenesis

Dysregulated cell-cell adhesion plays a critical role in epithelial cancer development. Studies of human and mouse cancers have indicated that loss of adhesion complexes known as adherens junctions contributes to tumor progression and metastasis. In contrast, little is known regarding the role of the related cell-cell adhesion junction, the desmosome, during cancer development. Studies analyzing expression of desmosome components during human cancer progression have yielded conflicting results, and therefore genetic studies using knockout mice to examine the functional consequence of desmosome inactivation for tumorigenesis are essential for elucidating the role of desmosomes in cancer development. Here, we investigate the consequences of desmosome loss for carcinogenesis by analyzing conditional knockout mice lacking Perp, a p53/p63 regulated gene that encodes an important component of desmosomes. Analysis of Perp-deficient mice in a UVB-induced squamous cell skin carcinoma model reveals that Perp ablation promotes both tumor initiation and progression. Tumor development is associated with inactivation of both of Perp's known functions, in apoptosis and cell-cell adhesion. Interestingly, Perp-deficient tumors exhibit widespread downregulation of desmosomal constituents while adherens junctions remain intact, suggesting that desmosome loss is a specific event important for tumorigenesis rather than a reflection of a general change in differentiation status. Similarly, human squamous cell carcinomas display loss of PERP expression with retention of adherens junctions components, indicating that this is a relevant stage of human cancer development. Using gene expression profiling, we show further that Perp loss induces a set of inflammation-related genes that could stimulate tumorigenesis. Together, these studies suggest that Perp-deficiency promotes cancer by enhancing cell survival, desmosome loss, and inflammation, and they highlight a fundamental role for Perp and desmosomes in tumor suppression. An understanding of the factors affecting cancer progression is important for ultimately improving the diagnosis, prognostication, and treatment of cancer.     

Solution to the phylogenetic enigma of Tetraplatia, a worm-shaped cnidarian

Tetraplatia is a genus containing two species of pelagic cnidarians of curious morphology. Their vermiform shape and four swimming flaps are difficult to relate to the features of other cnidarians, thus obscuring their phylogenetic affinities. Since their discovery in the mid-1800s, a number of prominent cnidarian workers have weighed in on this conundrum, some arguing that they are aberrant hydrozoans and others concluding that they are unusual scyphozoans. Current taxonomic practice conforms to the latter view. However, data presented here from the large and small subunits of the nuclear ribosome leave little doubt that Tetraplatia is in fact a hydrozoan genus. Indeed, its precise phylogenetic position is within Narcomedusae, as some authors had previously deduced based on structural characters. The distinctive body plan of Tetraplatia is remarkable because it appears to have a recent origin, in contrast to the prevailing pattern of metazoan history.     

31P-NMR of free and protein-bound molybdopterin guanine dinucleotide.

Molybdopterin guanine dinucleotide was studied by 31P-NMR in the free, iodoacetamide derivatized form [di(carboxamidomethyl)molybdopterin] and in the native state in the dimethyl sulfoxide reductase from Rhodobacter sphaeroides. The spectra confirm the presence of a pyrophosphate moiety in the cofactor molecule. Comparison of the spectrum of the free pterin with that of the protein-bound cofactor reveals a substantial upfield shift of the 31P resonances in the enzyme-bound form with respect to the free form. This shift is attributed to differences in the bond and torsional angles of the phosphates. The spectrum of the protein suggests significant coupling between the two phosphorus nuclei with coupling constants of approximately 200 Hz. Comparison of the 31P-NMR spectra of molybdopterin guanine dinucleotide and flavin adenine dinucleotide suggests that the two cofactors have similar conformations in both their free and protein-bound forms.     

Crystal structure of pyranose 2-oxidase from the white-rot fungus Peniophora sp

Pyranose 2-oxidase catalyzes the oxidation of a number of carbohydrates using dioxygen. The enzyme forms a D(2) symmetric homotetramer and contains one covalently bound FAD per subunit. The structure of the enzyme from Peniophora sp. was determined by multiwavelength anomalous diffraction (MAD) based on 96 selenium sites per crystallographic asymmetric unit and subsequently refined to good-quality indices. According to its chain fold, the enzyme belongs to the large glutathione reductase family and, in a more narrow sense, to the glucose-methanol-choline oxidoreductase (GMC) family. The tetramer contains a spacious central cavity from which the substrate enters one of the four active centers by penetrating a mobile barrier. Since this cavity can only be accessed by glucose-sized molecules, the enzyme does not convert sugars that are part of a larger molecule. The geometry of the active center and a comparison with an inhibitor complex of the homologous enzyme cellobiose dehydrogenase allow the modeling of the reaction at a high confidence level.     

Evolutionary relationships of flying foxes (genus Pteropus) in the Philippines inferred from DNA sequences of cytochrome b gene

Six flying fox species, genus Pteropus (four from the Philippines) were investigated using complete cytochrome b gene sequences (1140 bp) to infer their evolutionary relationships. The DNA sequences generated via polymerase chain reaction were analyzed using the neighbor-joining, parsimony, and maximum likelihood methods. We estimated that the first evolutionary event among these Pteropus species occurred approximately 13.90 ± 1.49 MYA. Within this short period of evolutionary time we further hypothesized that the ancestors of the flying foxes found in the Philippines experienced a subsequent diversification forming two clusters in the topology. The first cluster is composed of P. pumilus (Philippine endemic), P. speciosus (restricted in western Mindanao) with P. scapulatus, while the second one comprised P. vampyrus and P. dasymallus species based on the analysis from first and second codon positions. Consistently, all phylogenetic analyses divulged close association of P. dasymallus with P. vampyrus contradicting the previous report categorizing P. dasymallus under subniger species group with P. pumilus, P. speciosus, and P. hypomelanus. The Philippine endemic species (P. pumilus) is closely linked with P. speciosus. The representative samples of P. vampyrus showed a large genetic distance of 1.87%. The large genetic distance between P. dasymallus and P. hypomelanus, P. pumilus and P. speciosus denotes a distinct species group.     

Comparative Analysis of Different DNA Extraction Protocols: A Fast, Universal Maxi-Preparation of High Quality Plant DNA for Genetic Evaluation and Phylogenetic Studies

Four DNA extraction protocols were compared for ability to produce DNA from the leaves or needles of several species: oak, elm, pine, fir, poplar and maize (fresh materials) and rhododendron (silica dried or frozen material). With the exception of maize and poplar, the species are known to be difficult for DNA extraction. Two protocols represented classical procedures for lysis and purification, and the other two were a combination of classical lysis followed by anion exchange chromatography. The DNA obtained from all procedures was quantified and tested by PCR and Southern hybridisation.Test results indicated superiority of one of the four protocols; a combination of CTAB lysis followed by anion exchange chromatography which enabled DNA extraction from all seven species. A second protocol also produced DNA from leaves or needles of all species investigated and was well suited for PCR applications but not Southern hybridisations. The remaining protocols produced DNA from some but not all species tested.Abbreviations: CTAB, hexadecyltrimethylammonium bromide; EtOH, Ethanol; TBE, tris-borate-EDTA.     

The role of amino acid neurotransmitters in the descending control of electroreception

The roles of amino acid neurotransmitters in determining the processing characteristics of the electrosensory lateral line lobe (ELL) in Apteronotus leptorhynchus were investigated by studying the responses of ELL output neurons to pressure ejection of various neurotransmitter agonists and antagonists alone and in combination with simple electrosensory stimuli.

1. Pressure ejection of L-glutamate into the ELL dorsal molecular layer caused either excitation or inhibition of ELL efferent neurons (pyramidal cells). The sign of these responses reversed with changes in the position of the pressure pipette. Histological verification of drug ejection sites relative to recorded cells and diffusion estimates indicate that excitatory and inhibitory responses result from glutamate activation of pyramidal cells and of inhibitory interneurons, respectively.
2. ELL output cells respond to both NMDA and non-NMDA glutamate agonists and the responses are attenuated by co-ejection of specific antagonists indicating that both AMPA/kainate and NMDA receptors exist on pyramidal cell apical dendrites.
3. Gamma-aminobutyric acid inhibits basilar and nonbasilar pyramidal cells when ejected near their apical dendrites and disinhibits them when ejected near surrounding inhibitory interneurons confirming the presence of GABA receptors on these cell types.
4. An NMDA antagonist did not alter pyramidal cell responses to electrosensory stimuli but a non-NMDA antagonist altered both responses to the stimuli and firing frequency shortly following stimulus cessation.

     

The perennial biogas crops cup plant (Silphium perfoliatum L.) and field grass pose better autumn and overwintering habitats for arthropods than silage maize (Zea mays L.)

Perennial energy crops (PECs) can reduce the negative impacts of intensive silage maize cultivation on agroecosystems in Central Europe. Furthermore, the remaining vegetation of PECs after harvest may provide suitable habitat and more beneficial overwintering conditions for arthropods than maize. It was hypothesized that after harvest and in winter, arthropod abundance and biomass are higher in PECs than in silage maize. In a field experiment arranged in a factorial split-plot design of eight main plots (plot size: 240 m²), the two PECs cup plant (Silphium perfoliatum L.) and field grass were compared with silage maize (Zea mays L.) regarding their suitability as autumn (post-harvest) and overwintering habitats for arthropods. Soil temperature, moisture as well as biomass and abundance of autumn-active and overwintering arthropods were analyzed for these three crops. Suction sampling was used during autumn and emerging arthropods were sampled with emergence trap sets in spring. In PEC plots, soils were moister and less exposed to cold temperatures than in silage maize. Compared with silage maize, total arthropod abundance and biomass were higher in PEC plots for both sampling periods. Results were similar for most examined arthropod taxa. The results of this study demonstrate that, compared with silage maize, PECs provide suitable post-harvest habitats and constitute more suitable overwintering habitats for arthropods. Differences are likely to be based on lack of disturbance and the provision of vegetation structures after harvest that function as overwintering habitats for arthropods. It can be concluded that the positive effects of PECs on ground arthropods are not limited to their growing time but continue to a certain extend after harvest and during winter.