Design and synthesis of 4,5,6,7‐tetrahydro‐1H‐1,2‐diazepin‐7‐one derivatives as a new series of Phosphodiesterase 4 (PDE4) inhibitors

Phosphodiesterase 4 (PDE4) inhibitors have attractive therapeutic potential in respiratory, inflammatory, metabolic and CNS disorders. The present work details the design, chemical exploration and biological profile of a novel PDE4 inhibitor chemotype. A diazepinone ring was identified as an under-represented heterocyclic system fulfilling a set of PDE4 structure-based design hypotheses. Rapid exploration of the structure activity relationships for the series was enabled by robust and scalable two/three-steps parallel chemistry protocols. The resulting compounds demonstrated PDE4 inhibitory activity in cell free and cell-based assays comparable to the Zardaverine control used, suggesting potential avenues for their further development.     

Deciphering the structural role of histidine 83 for heme binding in hemophore HasA

Heme carrier HasA has a unique type of histidine/tyrosine heme iron ligation in which the iron ion is in a thermally driven two spin states equilibrium. We recently suggested that the H-bonding between Tyr75 and the invariantly conserved residue His83 modulates the strength of the iron-Tyr75 bond. To unravel the role of His83, we characterize the iron ligation and the electronic properties of both wild type and H83A mutant by a variety of spectroscopic techniques. Although His83 in wild type modulates the strength of the Tyr-iron bond, its removal causes detachment of the tyrosine ligand, thus giving rise to a series of pH-dependent equilibria among species with different axial ligation. The five coordinated species detected at physiological pH may represent a possible intermediate of the heme transfer mechanism to the receptor.     

Construction of a swine artificial chromosome: a novel vector for transgenesis in the pig

A de novo SAC was constructed by making use of YAC technology and a humanized yeast strain. The construct (about 310 kb) contained pig centromeric DNA and the Neo gene. The construct was introduced into a pig cell line by yeast-mammalian cell fusion and G418 resistant clones were obtained. One clone was characterized by FISH and shown to contain an episomally located microchromosome containing YAC, Neo and pig centromere sequences. FISH analysis over time showed that the SAC was mitotically stable for at least 34 generations in the absence of selection. The size of the SAC was determined by confocal microscopy of the SAC and shown to be approximately 7 Mb, which is about 25-fold greater than the size of the original YAC. From its behavior in pulsed field gel electrophoresis, FISH analysis of stretched DNA fibers, and its appearance under scanning confocal microscopy, it was concluded that the SAC is a circularized and multimerized derivative of the original YAC. Possible applications as vectors for animal transgenesis are discussed.     

Tremadoc (earliest Ordovician) brachiopods from Purmamarca and the Sierra de Mojotoro, Cordillera Oriental of northwestern Argentina

The Late Tremadoc storm-dominated shoreface to inner platform deposits exposed west of the Purmamarca village (Coquena Formation) contain a considerably more diverse brachiopod fauna than previously reported. Coquinite horizons from the lower heterolitic succession have yielded monospecific associations of Nanorthis purmamarcaensis nov. sp. (formerly assigned to N. christianiae KJERULF), which is also reported from the Late Tremadoc rocks of the Cerro San Bernardo area. The fine-grained Upper Member of the Coquena Formation contains a more diverse fauna composed by Nanorthis brachymyaria nov. sp., Astraborthis quebradensis nov. sp. and the new plectorthid genus Lipanorthis (type species L. andinus nov. sp.). A different species of Lipanorthis (L. santalaurae nov. sp.) from the Mid Tremadoc Floresta Formation of the Sierra de Mojotoro is also described.     

Long-term release of clodronate from biodegradable microspheres

This paper describes the formulation of a biodegradable microparticulate drug delivery system containing clodronate, a bisphosphonate intended for the treatment of bone diseases. Microspheres were prepared with several poly(D,L-lactide-co-glycolide) (PLGA) copolymers of various molecular weights and molar compositions and 1 poly(D,L-lactide) (PDLLA) homopolymer by a water-in-oil-in-water (w/o/w) double emulsion solvent evaporation procedure. Critical process parameters and formulation variables (ie, addition of stabilizing agents) were evaluated for their effect on drug encapsulation efficiency and clodronate release rate from microparticles Well-formed clodronate-loaded microspheres were obtained for all polymers by selecting suitable process parameters (inner water/oil volume ratio 1∶16, temperature-raising rate in the solvent evaporation step 1°C/min, 2% wt/vol NaCl in the external aqueous phase). Good yields were obtained in all batches of clodronate microspheres (above 60%); drug encapsulation efficiencies ranged between 49% and 75% depending on the polymer used. Clodronate release from all copolymer microspheres was completed in about 48 hours, while those from PDLLA microspheres required about 20 days. The change of microsphere composition by adding a surfactant such as Span 20 or a viscosing agent such as carboxymethylcellulose extended the long-term release up to 3 months. Clodronate was successfully entrapped in PLGA and PDLLA microspheres, and drug release could be modulated from 48 hours up to 3 months by suitable selection of polymer, composition, additives, and manufacturing conditions. Published: July 11, 2001.     

90S pre-ribosomes include the 35S pre-rRNA,the U3 snoRNP,and 40S subunit processing factors but predominantly lack 60S synthesis factors

We report the characterization of early pre-ribosomal particles. Twelve TAP-tagged components each showed nucleolar localization, sedimented at approximately 90S on sucrose gradients, and coprecipitated both the 35S pre-rRNA and the U3 snoRNA. Thirty-five non-ribosomal proteins were coprecipitated, including proteins associated with U3 (Nop56p, Nop58p, Sof1p, Rrp9, Dhr1p, Imp3p, Imp4p, and Mpp10p) and other factors required for 18S rRNA synthesis (Nop14p, Bms1p, and Krr1p). Mutations in components of the 90S pre-ribosomes impaired 40S subunit assembly and export. Strikingly, few components of recently characterized pre-60S ribosomes were identified in the 90S pre-ribosomes. We conclude that the 40S synthesis machinery predominately associates with the 35S pre-rRNA factors, whereas factors required for 60S subunit synthesis largely bind later, showing an unexpected dichotomy in binding.     

Temperature dependence of multiple high voltage activated Ca2+ channels in chick sensory neurones

The temperature dependence of high voltage activated Ca²⁺ channels has been investigated in cultured dorsal root ganglion neurones from chick embryos, using the cell-attached patch-clamp technique. The dihydropyridine sensitive L-type Ca²⁺ channel had a conductance of 23 pS, with 110 mM Ba²⁺ as charge carrier and in the presence of 3 M Bay K 8644. When the temperature was raised from 15 to 30 °C, the unitary channel current amplitude increased, with Q₁₀ value equal to 1.4. The rising phase of the averaged single-channel current became faster, with Q₁₀ value 2.7, whereas the decay phase showed a lower temperature sensitivity. Channel open probability decreased according to an exponential distribution of open and closed times. A second type of Ca²⁺ channel was identified, which was DHP-insensitive and had a lower conductance with a mean value equal to 13 pS. For the current amplitude, the Q₁₀ value was 1.3. Both activation and inactivation kinetics were strongly accelerated by an increase in temperature. The corresponding time constants gave Q₁₀ values equal to 5.9 for activation, and 2.0 for inactivation. Peak channel open probability was highly sensitive to a change in temperature, with a Q₁₀ value of 1.6. Finally, in -conotoxin GVIA pre-treated neurones, a non-inactivating DHP-insensitive Ca²⁺ channel with the lowest unitary conductance (10 pS) and a much lower temperature dependence was recorded. Single-channel current was increased by heating, with Q₁₀ value 1.3, whereas the channel kinetics were almost unaffected by temperature. Our data are consistent with the assumption that the different temperature dependence of the Ca²⁺ channel behaviours may be explained by separate gating processes of three types of Ca²⁺ channels.     

Effect of invader removal: pollinators stay but some native plants miss their new friend

Removal of invasive species often benefits biological diversity allowing ecosystems’ recovery. However, it is important to assess the functional roles that invaders may have established in their new areas to avoid unexpected results from species elimination. Invasive animal-pollinated plants may affect the plant–pollination interactions by changing pollinator availability and/or behaviour in the community. Thus, removal of an invasive plant may have important effects on pollinator community that may then be reflected positive or negatively on the reproductive success of native plants. The objective of this study was to assess the effect of removing Oxalis pes-caprae, an invasive weed widely spread in the Mediterranean basin, on plant–pollinator interactions and on the reproductive success of co-flowering native plants. For this, a disturbed area in central Portugal, where this species is highly abundant, was selected. Visitation rates, natural pollen loads, pollen tube growth and natural fruit set of native plants were compared in the presence of O. pes-caprae and after manual removal of their flowers. Our results showed a highly resilient pollination network but also revealed some facilitative effects of O. pes-caprae on the reproductive success of co-flowering native plants. Reproductive success of the native plants seems to depend not only on the number and diversity of floral visitors, but also on their efficiency as pollinators. The information provided on the effects of invasive species on the sexual reproductive success of natives is essential for adequate management of invaded areas.