Might the observed α2A-adrenoreceptor agonism or antagonism of allyphenyline analogues be ascribed to different molecular conformations?

We recently reported that the α(2)-adrenoreceptor (AR) ligand allyphenyline (9) significantly enhanced morphine analgesia (due to its α(2C)-AR agonism), was devoid of sedative side effects (due to its α(2A)-AR antagonism), prevented and reversed morphine tolerance and dependence. To highlight the molecular characteristics compatible with this behaviour and to obtain novel agents potentially useful in chronic pain and opioid addiction management, the allyl group of 9 was replaced by substituents of moderate steric bulk (MR) and positive or negative lipophilic (π) and electronic (σ) contributions in all the possible combinations. Effective novel α(2C)-agonists/α(2A)-antagonists (2, 3, 10, 12, and 17) were obtained. This study also demonstrated that contradictory combinations of the physicochemical parameters were similarly able to induce the α(2A)-activation. Since we had previously observed that the absolute configuration affected only the potency, but not the functional profile of the ligands, we hypothesized that the α(2A)-activation was governed by a ligand preferred conformation. From a structural overlay investigation it emerged that an extended conformation appeared to be associated with dual α(2C)-agonism/α(2A)-antagonism, whereas a folded conformation associated with α(2C)-/α(2A)-agonism.     

The relationship between vegetation density and its protective value depends on the densities and traits of prey and predators

Densities of submerged vegetation and those of associated animals tend to co‐vary. This relationship is often attributed to the positive correlation between the density of vegetation and its protective value against predators. However, two counteracting basic elements underlying this paradigm limit its generality. That is, increasing vegetation density should result in decreased predator–prey encounters, whilst at the same time predator–prey encounters should increase as animal densities increase. These two mechanisms should thus counteract each other when the densities of vegetation and associated animals, including both prey and predators, co‐vary. Experimental designs that expose fixed densities of prey and predators to varying densities of vegetation assess only the former mechanism and may thus not properly evaluate the protective value of vegetation in such conditions. By contrast, designs that mimic the naturally co‐varying organism densities test both mechanisms and thus their counteractive impacts on predator–prey encounters. We compared the outcomes of the two alternative designs and carried out additional experiments to explain the putative discrepancy. Increasing vegetation density (mimics of Potamogeton pectinatus) enhanced prey (Daphnia magna) survival only when fixed densities of prey and predators (Perca fluviatilis or Rutilus rutilus) were used. When the animal densities were allowed to co‐vary with vegetation density, vegetation had no impact on prey survival. Instead, prey survival was determined by the aggregate density of prey and predators, shaped by the species‐specific traits of the latter. Thus, the impact of the increased animal densities overrode the impact of the increased vegetation density on predator–prey encounters. It may be insufficient to attribute the co‐variation of vegetation, prey and predator densities simply to the association between vegetation density and its protective value. Increased food resources and reduced competition within vegetation may promote prey and thereby also predator abundance to a greater extent than previously thought.     

A gold-containing drug against parasitic polyamine metabolism: the X-ray structure of trypanothione reductase from Leishmania infantum in complex with auranofin reveals a dual mechanism of enzyme inhibition

Auranofin is a gold(I)-containing drug in clinical use as an antiarthritic agent. Recent studies showed that auranofin manifests interesting antiparasitic actions very likely arising from inhibition of parasitic enzymes involved in the control of the redox metabolism. Trypanothione reductase is a key enzyme of Leishmania infantum polyamine-dependent redox metabolism, and a validated target for antileishmanial drugs. As trypanothione reductase contains a dithiol motif at its active site and gold(I) compounds are known to be highly thiophilic, we explored whether auranofin might behave as an effective enzyme inhibitor and as a potential antileishmanial agent. Notably, enzymatic assays revealed that auranofin causes indeed a pronounced enzyme inhibition. To gain a deeper insight into the molecular basis of enzyme inhibition, crystals of the auranofin-bound enzyme, in the presence of NADPH, were prepared, and the X-ray crystal structure of the auranofin-trypanothione reductase-NADPH complex was solved at 3.5 ? resolution. In spite of the rather low resolution, these data were of sufficient quality as to identify the presence of the gold center and of the thiosugar of auranofin, and to locate them within the overall protein structure. Gold binds to the two active site cysteine residues of TR, i.e. Cys52 and Cys57, while the thiosugar moiety of auranofin binds to the trypanothione binding site; thus auranofin appears to inhibit TR through a dual mechanism. Auranofin kills the promastigote stage of L. infantum at micromolar concentration; these findings will contribute to the design of new drugs against leishmaniasis.     

CGHpower: exploring sample size calculations for chromosomal copy number experiments

Background  

Determining a suitable sample size is an important step in the planning of microarray experiments. Increasing the number of arrays gives more statistical power, but adds to the total cost of the experiment. Several approaches for sample size determination have been developed for expression array studies, but so far none has been proposed for array comparative genomic hybridization (aCGH).     

alpha 2B-Adrenoceptor levels govern agonist and inverse agonist responses in PC12 cells

Receptor density is an important determinant of cellular effector responses to receptor activation. We analysed cytosolic Ca(2+) responses to alpha(2)-adrenergic agents in PC12 cells expressing human alpha(2B)-adrenergic receptors (AR) at two densities (3.8 and 1.3 pmol/mg protein). The efficacy (E(max)) of agonists was greater in cells with higher receptor expression; while the potency (EC(50)) of norepinephrine and oxymetazoline was independent of alpha(2B)-AR levels. Several classical alpha(2)-AR antagonists behaved as either partial or inverse agonists in a receptor density-dependent fashion. No apparent structural similarities were found among the inverse agonists, precluding simple predictions of inverse agonist activity. Transfected PC12 cells expressing alpha(2B)-AR at relatively high density would be a useful approach to screen inverse agonists for this class of receptors. Our results further indicate that receptor density significantly influences the properties of ligands, not only of partial agonists as predicted by classical receptor theory, but also of antagonists and full agonists.     

Escherichia coli flavohemoglobin is an efficient alkylhydroperoxide reductase

Escherichia coli flavohemoglobin (HMP) is shown to be capable of catalyzing the reduction of several alkylhydroperoxide substrates into their corresponding alcohols using NADH as an electron donor. In particular, HMP possesses a high catalytic activity and a low Km toward cumyl, linoleic acid, and tert-butyl hydroperoxides, whereas it is a less efficient hydrogen peroxide scavenger. An analysis of UV-visible spectra during the stationary state reveals that at variance with classical peroxidases, HMP turns over in the ferrous state. In particular, an iron oxygen adduct intermediate whose spectrum is similar to that reported for the oxo-ferryl derivative in peroxidases (Compound II), has been identified during the catalysis of hydrogen peroxide reduction. This finding suggests that hydroperoxide cleavage occurs upon direct binding of a peroxide oxygen atom to the ferrous heme iron. Competitive inhibition of the alkylhydroperoxide reductase activity by carbon monoxide has also been observed, thus confirming that heme iron is directly involved in the catalytic mechanism of hydroperoxide reduction. The alkylhydroperoxide reductase activity taken together with the unique lipid binding properties of HMP suggests that this protein is most likely involved in the repair of the lipid membrane oxidative damage generated during oxidative/nitrosative stress.     

Tyrosinase-mediated quinone tanning of chitinous materials

Stable and self-sustaining gels were obtained from tyrosine glucan (a modified chitosan synthesized with 4-hydroxyphenylpyruvic acid) in the presence of tyrosinase. Similar gels were obtained from 3-hydroxybenzaldehyde, 4-hydroxybenzaldehyde and 3,4-dihydroxybenzaldehyde: all of them were hydrolyzed by lysozyme, lipase and papain. Microcapsules were similarly obtained by introducing tyrosinase in a water-in-oil emulsion containing tyrosine glucan in the water phase. No cross-linking was observed for chitosan derivatives of vanillin, syringaldehyde and salicylaldehyde. Collagen-chitosan-tannin mixtures were also studied under the catalytic action of tyrosinase: partially crystalline, hard, mechanically resistant and scarcely wettable materials were obtained upon drying. By contrast, products obtained from albumin, pseudocollagen and gelatin, in the presence of a number of phenols and chitosan under comparable conditions, were brittle.     

Simultaneous determination of 3-methoxy-4-hydroxyphenylglycol, 5-hydroxyindoleacetic acid, and homovanillic acid in cerebrospinal fluid with high-performance liquid chromatography using electrochemical detection

An improved high-performance liquid chromatographic method with electrochemical detection (HPLC-EC) for the simultaneous determination of 3-methoxy-4-hydroxyphenylglycol (MHPG), 5-hydroxyindoleacetic acid (5-HIAA), and homovanillic acid (HVA) in cerebrospinal fluid (CSF) of humans and nonhuman primates is described. Quantitation is based on the use of an internal standard, 5-fluoro-HVA. Sample preparation consists of mixing an aliquot of CSF with a solution of the internal standard followed by ultrafiltration. The precision of the method is high, with within-run and between-run coefficients of variation of 2-6% and less than 10%, respectively, in the concentration ranges of the metabolites encountered in human lumbar CSF. Accuracy was tested by comparing the present HPLC method with specific gas chromatographic-mass spectrometric (GS-MS) assays for MHPG and HVA and a GC-MS-validated HPLC assay for 5-HIAA: the correlations obtained were 0.968 for MHPG, 0.989 for 5-HIAA, and 0.999 for HVA, with no systematic bias between the methods. The use of ascorbate as a preserving agent for monoamine metabolites in CSF was not found to be necessary when proper care was exercised in sample handling and storage. The analysis of samples with up to 2% ascorbic acid was possible as well, but MHPG had to be assayed separately using an extraction procedure and an alternative internal standard, 3-ethoxy-4-hydroxyphenylglycol.     

Neurochemical and behavioural changes in zebrafish Danio rerio after systemic administration of 6-hydroxydopamine and 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine

Dopaminergic deficiency in the brain of zebrafish was produced by systemic administration of two catecholaminergic neurotoxins, 6-hydroxydopamine (6-OHDA) and 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP), and the neurochemical and behavioural changes were characterized. The levels of dopamine and noradrenaline decreased significantly after the injection of MPTP and 6-OHDA. Corresponding to these changes, fish exhibited characteristic changes in locomotor behaviour, i.e. the total distance moved and velocity decreased after both neurotoxins. Tyrosine hydroxylase and caspase 3 protein levels were not altered after MPTP or 6-OHDA injections, as studied by immunohistochemistry and western blotting. The catecholaminergic cell clusters suggested to correspond to the mammalian nigrostriatal cell group displayed normal tyrosine hydroxylase immunoreactivity after the toxin treatment and did not show signs of DNA fragmentation that would indicate activation of cascades that lead to cell death. The results show that single systemic injections of MPTP and 6-OHDA induce both biochemical and behavioural changes in zebrafish, albeit failing to produce any significant morphological alteration in catecholaminergic cell clusters at the tested doses. This approach may be used for the screening of chemicals affecting the dopaminergic system. The model may be especially useful for evaluation of the role of novel genes in neurotoxicity, as a large number of zebrafish mutants are becoming available.     

Uneven cellular expression of recombinant alpha2A-adrenoceptors in transfected CHO cells results in loss of response in adenylyl cyclase inhibition

Two populations of Chinese hamster ovary (CHO) cells expressing similar numbers of recombinant human alpha2A-adrenergic receptors (alpha2A-AR) showed different capacity to inhibit adenylyl cyclase (AC) activity. Cells transfected with an integrating vector exhibited agonist-dependent inhibition of forskolin-stimulated AC, whereas cells transfected with a non-integrating episomal vector showed no inhibition. Fluorescent microscopy and flow cytometry revealed a very uneven receptor distribution in the episomally transfected cell population. Monoclonal cell populations were expanded from this parent population. Most clones lacked significant amounts of receptors, while a few expressed receptors at high density; these exhibited efficient agonist-dependent inhibition of forskolin-stimulated AC activity. Thus, dense receptor expression in only a few cells is not sufficient to evoke a significant inhibitory response in a functional assay where AC is stimulated in all cells. Consequently, a false negative result was produced. Furthermore, the cell population transfected with an integrating vector showed loss of homogeneity with increasing passage number.