Characterisation of the Binding of [3H]WAY-100635, a Novel 5-Hydroxytryptamine1A Receptor Antagonist, to Rat Brain

Abstract: The specific binding of [³H]WAY-100635 {N-[2-[4-(2-[O-methyl-³H]methoxyphenyl)-1-piperazinyl]ethyl]-N-(2-pyridinyl)cyclohexane carboxamide trihydrochloride} to rat hippocampal membrane preparations was time, temperature, and tissue concentration dependent. The rates of [³H]WAY-100635 association (k₊₁ = 0.069 ± 0.015 nM^?1 min^?1) and dissociation (k_?1 = 0.023 ± 0.001 min^?1) followed monoexponential kinetics. Saturation binding isotherms of [³H]WAY-100635 exhibited a single class of recognition site with an affinity of 0.37 ± 0.051 nM and a maximal binding capacity (B_max) of 312 ± 12 fmol/mg of protein. The maximal number of binding sites labelled by [³H]WAY-100635 was ～36% higher compared with that of 8-hydroxy-2-(di-n-[³H]-propylamino)tetralin ([³H]8-OH-DPAT). The binding affinity of [³H]WAY-100635 was significantly lowered by the divalent cations CaCl₂ (2.5-fold; p < 0.02) and MnCl₂ (3.6-fold; p < 0.05), with no effect on B_max. Guanyl nucleotides failed to influence the K_D and B_max parameters of [³H]WAY-100635 binding to 5-HT_1A receptors. The pharmacological binding profile of [³H]WAY-100635 was closely correlated with that of [³H]8-OH-DPAT, which is consistent with the labelling of 5-hydroxytryptamine_1A (5-HT_1A) sites in rat hippocampus. [³H]WAY-100635 competition curves with 5-HT_1A agonists and partial agonists were best resolved into high- and low-affinity binding components, whereas antagonists were best described by a one-site binding model. In the presence of 50 µM guanosine 5′-O-(3-thiotriphosphate) (GTPγS), competition curves for the antagonists remained unaltered, whereas the agonist and partial agonist curves were shifted to the right, reflecting an influence of G protein coupling on agonist versus antagonist binding to the 5-HT_1A receptor. However, a residual (16 ± 2%) high-affinity agonist binding component was still apparent in the presence of GTPγS, indicating the existence of GTP-insensitive sites.     

Conformation of cyclobradykinin by nmr and distance geometry calculations

The conformation of the head-to-tail cyclic analogue of bradykinin in DMSO was investigated by nmr. Three sets of resonances were detected and fully assigned. These were attributed to the presence of three stable conformers, two of which were exchanging on the nmr time scale. A fourth, incomplete set of resonances was detected but not assigned. The three major conformers differ in the conformation at the three X-Pro bonds present. From nuclear Overhauser effect spectroscopy (NOESY) spectra, three sets of interproton distances were derived and used in NOE-restrained distance geometry calculations. The resulting structures were refined by energy minimization to yield families of structures. Conformer I is characterized by the presence of two type VIb β-turns between Arg¹ and Gly⁴ and between Phe⁵ and Phe⁸. The first β-turn is present also in conformer II, while an inverse γ-turn bridging Pro³ is the most pronounced structural feature of conformer III. © 1995 John Wiley & Sons, Inc.     

Operational patterns affecting lactic acid production in ultrafiltration cell recycle bioreactor

Lactic acid production with cell recycling on an ultrafiltration tubular membrane reactor was studied; higher lactic acid concentrations as well as productivities were obtained under long-term fermentations compared with other high cell density systems. Different operational conditions, namely dilution rates and start-up modes, were assessed. Performances were very different at the three different dilution rates tested (D = 0.20 h(-1), D = 0.40 h(-1), or D = 0.58 h(-1)). The different behaviours are discussed and factors responsible for them are presented. The best way to operate for lactic acid production is chosen, the dilution rate of D = 0.40 h(-1) being the one providing the best overall performance. On the other hand, results show that of the two start-up modes tested, continuous start (membrane open) permits higher permeabilities throughout the operational runs than batch start (membrane closed). Operational stability was found to be directly associated with membranes that work at "steady state," the membrane permeability being kept around 15 L/m(2) h. Optimized cell bleed can improve time of operation if such membrane permeability can be maintained for a longer time. A comparison of results with those obtained in other lactic acid production systems is presented; such comparison shows that this tubular ultrafiltration membrane cell recycle reactor presents three important advantages: (1) concomitant lactic acid concentrations and productivities; (2) long periods of operation at reasonable permeabilities; and (3) good mechanical stability permitting the use of steam sterilization. (c) 1995 John Wiley & Sons, Inc.     

Anaerobic digestion of Ulva sp. 1. Relationship between Ulva composition and methanisation

Ulva often represents the main component of mass algal growths, and its composition and degradability make it a relatively good methanisation substrate. In ‘green tides’ Ulva sp. from Brittany, the low content oflignin-type components (polyphloroglucinols: 1.3% dry weight), and the large hemicellulosic fraction (9% dry weight) favour the substrate's accessibility to enzymes. Anaerobic degradation with a batch orcompletely stirred system is technically possible. However, the methane yield reached only 0.20 m³ kg⁻¹ volatile solids and the epuration rate 50% volatile solids in experiments in batch or completely stirred reactors. More generally, methanisation comes up against various practical obstacles: seasonal growth of Ulva, low density of alga in suspension for loading the digester, high S concentration leading to the production of a biogas with a high H2S content, and, finally, the existence of a refractory or slowly degradable part, which requires a compromise between productivity and biological yield. This revised version was published online in August 2006 with corrections to the Cover Date.     

Hyperbolic Modeling of Starburst Dendrimers

Starburst dendrimers are highly branched oligomers. A rigid dendritic hydrocarbon, C₁₁₃₄H₁₁₄₆, has recently been synthesized. It consists of 94 phenylacetylene units displayed in a self-similar two-dimensional skeleton isomorphous to the three-coordinated Bethe lattice. The three-dimensional representation of phenylacetylene dendrimer shows a globular architecture with large voids and niches in its interior, characteristic of hyperbolic surfaces. This work investigates the geometrical scaling behavior of this starburst dendrimer using the symmetry properties of a Bethe lattice embedded in the hyperbolic plane. The results for C₁₁₃₄H₁₁₄₆ provide its density profile and an upper bound for its macromolecular size. This revised version was published online in June 2006 with corrections to the Cover Date.     

Protracted release of the LHRH agonist avorelin (MF 6001) from two depot formulations in dogs and men

Avorelin is a new superagonist of naturalluteinizing-hormone-releasing-hormone. Avorelin hasbeen formulated in high molecular weight polylactic glycolic acid to afford protracted andcontinuous release of the peptide from subcutaneousimplants. Two different formulations (10 and 15 mg)were tested first in dogs and then in men during aclinical phase II trial. Chemical castration wasmaintained for at least 6 months in dogs withboth formulations. A similar duration of activity(approximately 6 months) was observed in men.     

The membrane-bound high-molecular-mass cytochromes c from Desulfovibrio gigas and Desulfovibrio vulgaris Hildenborough; EPR and Mössbauer studies

The high-molecular-mass cytochromes c (Hmcs) from the sulfate-reducing bacteria Desulfovibrio gigas and Desulfovibrio vulgaris (Hildenborough) were found to be strongly bound to the cytoplasmic membrane. After detergent solubilization they were shown to be water soluble and to be similar to those previously isolated from the soluble fractions in terms of N-terminal sequence, molecular mass, UV-visible and EPR spectroscopies. In D. gigas, higher amounts of Hmc can be obtained from the membranes than from the soluble fraction. This enabled further characterization of both cytochromes. The apparent heme reduction potentials of both Hmcs, determined at pH 7.5 through visible and EPR redox titrations, span a large range of redox potentials, approximately between 0 and –280?mV, and can be roughly divided into three groups: four to five hemes have E ⁰s of –30?mV to –100?mV, three to four hemes have E ⁰s around –170?mV, and seven to eight hemes have a lower E ⁰ of –250 to –280?mV. Several of these redox potentials are strongly pH dependent. Mössbauer studies of oxidized and reduced D. vulgaris Hmc show that this protein contains two high-spin hemes in both oxidation states. The rate of reduction of both Hmcs with the periplasmic hydrogenases from the corresponding organisms is extremely slow.     

Hydrological cycle and interannual variability of the aquatic community in a temporary saline lake (Fuente de Piedra,Southern Spain)

Fuente de Piedra is a shallow, temporary saline lake whoseseasonal behavior is strongly dependent on the annual hydrologicalbudget. In this study, we outline the characteristics of Fuente dePiedra Lake for two years that had different hydrological budgets.The high precipitations in 1989–90 caused the lake not to dry asusual, and decreased both salinity and the amplitude of changes.There were also differences in nutrient dynamics, with generallylower concentrations of soluble reactive phosphorus and ammonium,whereas in the more humid year nitrate showed a distinct maximum inwinter. Winter bloom chlorophyll a concentrations were alsomuch higher in 1989–90 (>600 g l-1) but there wasalso a winter productive phase that was presumably poorly coupledwith consumption processes that predominate in spring. Planktonicassemblages were different between years. Highly halotolerantphytoplankton species (Dunaliella salina and D. viridis) became scarcer, and especially two previouslyunrecorded diatoms (Cyclotella sp. and Chaetoceros sp.)became dominant in the bloom time in the wet year. The speciesrichness of the zooplankton increased in the wet year, with newspecies appearing that were not collected during 1987–88(Branchinectella media, Daphnia mediterranea, Macrothrix sp.,Arctodiaptomus salinus, Cyclops sp., Sigara sp...).There was also a much higher development of macrophytes (Ruppiadrepanensis, Althenia orientalis, Lamprothamnium papulosum)and bird populations, especially flamingoes (Phoenicopterusruber).Important interannual variations in this sort of system pointto the need for long term studies to observe the whole range ofstates that define the lake as an entity.     

A non-linear model of phosphorus flux in the phytoplankton of a temperate eutrophic reservoir

Phosphorus fluxes in phytoplankton, between intracellular andexternal compartments have been measured, in the epilimnion ofatemperate eutrophic reservoir, by means of phosphorusdepletionexperiments performed in situ. Flow rates betweencompartments were plotted against the phosphorus concentrationofthe compartments' origin of the flows and fitted to saturationfunctions.A value of phosphorus cell subsistence quota for the wholecommunity has been computed from the X-intercept values ofeveryfunction. The obtained figures are 10–100 times higher,dependingon the reference value, than those measured running theclassicalsingle species growth response experiments in chemostats.We propose a model using five compartments and a series offlowrates between them in order to simulate the general phosphoruscycling in freshwater phytoplankton. Short term simulation ofphosphorus content in every compartment shows a fluctuatingpatternaround rates of equilibrium, in agreement with the pattern ofvariation observed in situ for the selectedcompartments.