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Abstract: The kinetic data obtained from the action of a cathepsin D-like enzyme from Biomphalaria glabrata hepatopancreas (digestive gland) on MOCAc-Gly-Lys-Pro-Ile-Leu-Phe-Phe-Arg-Leu-Lys(DNp)-D–Arg-NH2, was studied as a data prototype, generated by means of a fluorogenic substrate. An initial fluorescence, due to incomplete energy transfer, of about 8% of the values attained after complete substrate hydrolysis; a non-linear standard curve even at μM concentrations and an exponential decay of the steady state fluorescence of reaction product of the order of 10−4 ×s−1 were the main analytical problems encountered. The standard curves for fluorescence of the substrate reaction product after 48 h of hydrolysis, and the reference compound MOCAc-Pro-Leu-Gly-NH2, were fitted by polynomial approximation and the point derivates used as calibration factors. Time dependence of the calibration factor for the reaction product was −2.96×10−4 a.u μM−1 ×s−1 that is, in the same order of observed enzymic reaction rates. A mathematical treatment was devised for obtaining rates corrected for errors derived from the three analytical problems indicated. The method is of general application in continuous fluorometric assays, irrespective of the particular enzyme used, but of special value for substrates that present significant initial fluorescence. The reaction rates were 11% higher; as calculated by means of the calibration factor [substrate]÷(final−initial fluorescence intensities), which is the prevalent procedure in the literature; leading to underestimation of K m and overestimation of V max. [Copyright &y& Elsevier]

Abstract: The amygdala has emerged as an important forebrain modulator of arousal. Acetylcholine plays a role in the regulation of sleep and wakefulness, particularly rapid eye movement sleep (REM). The major cholinergic input to the amygdala comes from the basal forebrain, a region primarily linked to wakefulness. We examined sleep and the encephalogram for 8 h following bilateral microinjections into the central nucleus of the amygdala (CNA) of the cholinergic agonist, carbachol (CARBL: 0.3 μg; CARBH: 3.0 μg), the acetylcholinesterase inhibitor, neostigmine (NEOL: 0.3 μg; NEOH: 3.0 μg), the muscarinic antagonist, scopolamine (SCOL: 0.3 μg; SCOH: 1.0 μg), the nicotinic antagonist, mecamylamine (MECL: 0.3 μg; MECH: 1.0 μg) and saline (SAL, 0.2 μl) alone. Both doses of CARB and NEO significantly reduced REM, but did not significantly alter non-rapid eye movement sleep (NREM). Both doses of SCO significantly increased NREM, and SCOH also produced an initial increase in REM followed by a significant decrease. CARBH and NEOH decreased REM electroencephalogram (EEG) power in the 5.5–10 Hz band, and NEOL and NEOH decreased NREM EEG power in the 0.5–5.0 Hz band. CARBL decreased waking EEG power in the 0.5–5.0 Hz band, and NEOH decreased waking EEG power in the 5.0–10.0 Hz band. Both doses of SCO significantly increased waking EEG power in the 5.5–10.0 Hz band. Compared with SAL, MEC did not significantly alter sleep or EEG power. The reduction of REM by CARB and NEO and the alteration of sleep by SCO indicate that cholinergic regulation of the amygdala is involved in the control of arousal in rodents. In contrast, CARB microinjections into CNA increase REM in cats, though the reasons for the species difference are not known. The results are discussed in the context of anatomical inputs and species differences in the cholinergic regulation of CNA. [Copyright &y& Elsevier]

Abstract: The present study was designed to evaluate the effects of novel and recognised compounds at human recombinant A2B adenosine receptors expressed in Chinese hamster ovary (hA2BCHO), in human embryonic kidney 293 (hA2BHEK-293) and at endogenous A2B receptors in human mast cells (HMC-1). Saturation binding experiments performed using the new high affinity A2B adenosine radioligand [3H]-N-benzo[1,3]dioxol-5-yl-2-[5-(2,6-dioxo-1,3-dipropyl-2,3,6,7-tetra hydro-1H-purin-8-yl)-1-methyl-1H-pyrazol-3-yloxy]-acetamide ([3H]-MRE 2029F20) revealed a single class of binding sites in hA2BCHO, hA2BHEK-293 and HMC-1 cells with K D (nM) of 1.65±0.18, 2.83±0.34, 2.62±0.27 and B max (fmol/mg protein) of 36±4, 475±50 and 128±15, respectively. The pharmacological profile of new compounds, determined in inhibition binding experiments in hA2BHEK-293 cells using [3H]-MRE 2029F20, showed a rank order of potency typical of the A2B receptors with K i values in the range 3.2–28nM. In functional assays, recognised agonists and antagonists were studied by evaluating their capability to modulate the cAMP production in hA2BCHO and in HMC-1 cells. Novel compounds were able to decrease NECA-stimulated cAMP production in hA2BCHO and in HMC-1 cells showing a high potency. New compounds were also able to inhibit cAMP levels in the absence of NECA and in the presence of forskolin stimulation in hA2BCHO and in HMC-1 cells. In HEK-293 cells MRE 2029F20 reduced cAMP basal levels with an IC50 value of 2.9±0.3nM. These results suggest that novel compounds are antagonists with an inverse agonist activity in recombinant and native human A2B receptors. [Copyright &y& Elsevier]

Abstract: 1,3-Butadiene and isoprene (2-methyl-1,3-butadiene) are chemically related substances that are carcinogenic to rodents. The overall aim of this work is to elucidate the role of the genotoxic action of diepoxide metabolites in the carcinogenesis of the dialkenes. In vivo doses of the diepoxide metabolites were measured through reaction products with hemoglobin (Hb adducts) in studies of induced micronuclei (MN) in rodents. In the reaction with N-terminal valine in Hb, diepoxybutane and isoprenediepoxide form ring-closed adducts, pyrrolidines [N,N-(2,3-dihydroxy-1,4-butadiyl)valine and N,N-(2,3-dihydroxy-2-methyl-1,4-butadiyl)valine, respectively]. The method applied for Hb-adduct measurement is based on tryptic degradation of the protein and liquid chromatography electrospray ionisation tandem mass spectrometry (LC–ESI-MS/MS) analysis. Mice were given single i.p. injections of the monoepoxides of butadiene and isoprene, 1,2-epoxy-3-butene or 1,2-epoxy-2-methyl-3-butene, respectively. Rats were treated in the same way with 1,2-epoxy-3-butene. In mice pyrrolidine adduct levels increased with increasing administered doses of the monoepoxides. The in vivo dose of diepoxybutane was on average twice as high (0.29±0.059mMh) as the in vivo dose of isoprenediepoxide (0.15±0.053mMh) per administered dose (mmol/kg body weight) of the monoepoxides. In mice the genotoxic effects of the two monoepoxides, measured as the increase in the frequencies of micronuclei (MN), were approximately linearly correlated to the in vivo doses of the diepoxides (except at the highest dose of diepoxybutane). In rats the pyrrolidine-adduct levels from diepoxybutane were below the limit of quantification at all administered doses of 1,2-epoxy-3-butene and no significant increase was observed in the frequency of MN. Measurement of the ring-closed adducts to N-termini in Hb by the applied method permits analysis of in vivo doses of diepoxybutane and isoprenediepoxide, which may be further used for the elucidation of the mechanisms of carcinogenesis of butadiene and isoprene. [Copyright &y& Elsevier]

Abstract: Solution studies on the binding properties of N-2,4-dimethylphenyl-N′-ethylformamidine (amitraz) toward nine lanthanide ions including lanthanum, cerium, neodium, samarium, europium, gadolinium, terbium, dysprosium, ytterbium and some other transition and heavy metal ions such as copper, lead, cobalt, nickel ions, showed a selective 1:1 complexation between amitraz and lanthanum ions. Consequently, amitraz was applied as an ion carrier in construction of a novel poly(vinyl chloride) membrane sensor for La(III). The sensor has a linear dynamic range of 1.0×10-1 to 1.0×10-7M with a Nernstian slope of 19.8±0.2mV per decade and a detection limit of 8.0×10-8M. The proposed sensor displays a fast response time (<8s), and can be used for at least 2 months without any considerable divergences in the potentials. The La(III) membrane sensor revealed comparatively good selectivity with respect to most of cations including alkaline, alkaline earth, and some transition and heavy metal ions. It could be used in a pH range of 3.0–9.0. The proposed membrane electrode was used as an indicator electrode in the potentiometric titration of La(III) ions with an EDTA solution, and also in the determination of fluoride concentration in some mouth wash preparations. [Copyright &y& Elsevier]