Rapid Communication: cis-Methyldioxolane Specifically Recognizes the m2 Muscarinic Receptor

Abstract: cis -Methyldioxolane (CD) is a muscarinic receptor agonist. [³H] CD has been used to label a subpopulation of muscarinic receptors described as exhibiting high agonist affinity. Pharmacological evidence suggests that the population of receptors labeled by [³H] CD consists of m2 and/or m4 subtypes; however, no studies have directly addressed the subtype selectivity of [³H] CD. The present study characterizes binding of this ligand to individual human receptor subtypes expressed in transfected Chinese hamster ovary cells. Results indicate that [³H] CD binds with high affinity only to Hm2 receptors but not to all Hm2 receptors. Twenty-eight percent of Hm2 receptors bound [³H] CD with a K _D of 3.5 ± 0.5 nM. Binding was eliminated in the presence of guanosine 5'- O -(3-thiotriphosphate), indicating that the Hm2 receptors labeled by [³H] CD are those that are associated with GDP-bound G protein. Binding of [³H] CD by only a subpopulation of Hm2 receptors is in agreement with data generated from studies of [³H] CD binding in mammalian brain. Because muscarinic receptors have been implicated to play a role in the pathogenesis of both Alzheimer's and Parkinson's disease, as well as the neurotoxicity of organophosphorus compounds, knowledge of the binding specificity of the muscarinic agonist [³H] CD should aid research in these areas.     

In vitro binding of [14C]2,5-hexanedione to rat neuronal cytoskeletal proteins

2,5-Hexanedione (2,5-HD) induces central-peripheral axonpathy characterized by the accumulation of 10-nm neurofilaments proximal to the nodes of Ranvier and a Wallerian-type degeneration. It has been postulated that neurofilament crosslinking may be involved in the production of this axonopathy. A potential initiating event in this neurotoxic process may be the direct binding of 2,5-HD to neurofilament and microtubule proteins. In this study, the in vitro binding of [¹⁴C]2,5-HD to neurofilament and microtubule proteins was examined. Neurofilament proteins isolated from rat spinal cord or microtubule proteins isolated from rat brain were incubated in the presence of 2,5-HD at concentrations ranging 25 to 500 mM. Quantitative analysis of sodium dodecyl sulfate (SDS) polyacrylamide gels revealed a dose- and time-dependent binding of 2,5-HD to both neurofilament proteins and microtubule proteins. Expressed as pmol 2,5-HD bound per g protein, the observed relative binding was MAP2>NF160>NF200>NF68>tubulin. These data demonstrate the direct binding of 2,5-HD to cytoskeletal proteins including both neurofilaments and microtubules.     

Purification and characterization of cytochrome P450 isozymes from beta-naphthoflavone-induced adult hen liver

Cytochromes P450 beta NF-A, beta NF-B, and beta NF-C were purified from beta-naphthoflavone-treated adult hens. Cytochrome P450 beta NF-A, however, appeared at two places in the purification scheme. They were designated as cytochromes P450 beta NF-A1 and beta NF-A2 for property comparison. The cytochromes beta NF-A1 and beta NF-A2 were induced by both phenobarbital and beta-naphthoflavone treatment and were similar to P450 PB-A (previously purified from phenobarbital-induced hen livers) in molecular weights, isoelectric pH, spectral properties, behavior on chromatography columns, catalysis of substrates, immunological cross-reactivity on Ouchterlony plates and by immunoblotting, and NH2-terminal amino acid sequence. However, P450 PB-A differed from beta NF-A1/beta NF-A2 in peptide pattern after partial proteolysis by alpha-chymotrypsin and Staphylococcus aureus V8 protease, and complete digestion of 125I-labeled cytochromes by trypsin. The cytochrome P450 PB-A also differed from beta NF-A1/beta NF-A2, in that its antibodies cross-reacted with P-450 of normal, PB-, and beta-NF-induced rabbit liver microsomes. The cytochromes beta NF-B and beta NF-C, although immunochemically cross-reactive with each other, were distinct enzymes on the basis of molecular weights, spectral characteristics, isoelectric pH, peptide pattern on partial proteolysis, tryptic peptide pattern, cross-reactivity of their antibodies with other species, and NH2-terminal amino acid sequence. The most notable difference between beta NF-B and beta NF-C was that the anti-beta NF-C IgG completely inhibited O-dealkylation of 7-methoxyresorufin and 7-ethoxyresorufin by beta-NF-induced microsomes. These activities increased 40- to 50-fold in beta-NF-induced microsomes as compared to only 2- to 4-fold in PB-treated hens. The amino-terminal sequences of beta NF-B and beta NF-C were different from those of mammalian and other nonmammalian species.     

Kinetic measurements of the release of prostaglandins from perfused rat lungs

Prostaglandins released from isolated, ventilated and perfused rat lungs were measured by a simple modification of the Vane technique using the rat stomach fundus as a continuous bioassay tissue. Exogeneously supplied arachidonic acid was converted mainly to PGF_2α which was determined by bioassay. A novel method for mixing a stream of inhibitors with the perfusate was used to determine PGF_2α in the presence of substrate amounts of arachidonic acid. Using this system the apparent K_m for PGF_2α production with arachidonic acid as the substrate was found to be 1.90 × 10⁻⁴M, while the K_i for aspirin was found to be 2.47 × 10⁻⁴M. These kinetic parameters are close to those reported for cell free systems and subcellular fractions suggesting that both substrate and inhibitor have ready access to the site of prostaglandin synthesis. The method appears to be generally useful to determine the effect of drugs and environmental factors on the release of prostaglandins by the lung.     

Affinity of Ionic Species of Nucleoside Transport Protein Inhibitors

Abstract

A class of very potent nucleoside transport inhibitors is present in two molecular forms around physiological pH. We investigated whether the monoprotonated or the unionized species of these molecules binds to this camer protein with higher affinity.     

THE EFFECTS OF INJURY PREVENTION WARM-UP PROGRAMMES ON KNEE STRENGTH IN MALE SOCCER PLAYERS

The study investigates the effects of the 11+ and HarmoKnee injury prevention programmes on knee strength in male soccer players. Under-21-year-old players (n=36) were divided equally into: the 11+, HarmoKnee and control groups. The programmes were performed for 24 sessions (20-25 min each). The hamstrings and quadriceps strength were measured bilaterally at 60°·s^-1, 180°·s^-1 and 300°·s^-1. The concentric quadriceps peak torque (PT) of the 11+ increased by 27.7% at 300°·s^-1 in the dominant leg (p<0.05). The concentric quadriceps PT of HarmoKnee increased by 36.6%, 36.2% and 28% in the dominant leg, and by 31.3%, 31.7% and 20.05% at 60°·s^-1, 180°·s^-1 and 300°·s^-1 in the non-dominant leg respectively. In the 11+ group the concentric hamstring PT increased by 22%, 21.4% and 22.1% at 60°·s^-1, 180°·s^-1 and 300°·s^-1, respectively in the dominant leg, and by 22.3%, and 15.7% at 60°·s^-1 and 180°·s^-1, in the non-dominant leg. In the HarmoKnee group the hamstrings in the dominant leg showed an increase in PT by 32.5%, 31.3% and 14.3% at 60°·s^-1, 180°·s^-1 and 300°·s^-1, and in the non-dominant leg hamstrings PT increased by 21.1% and 19.3% at 60°·s^-1 and 180°·s^-1 respectively. The concentric hamstrings strength was significantly different between the 11+ and control groups in the dominant (p=0.01) and non-dominant legs (p=0.02). The HarmoKnee programme enhanced the concentric strength of quadriceps. The 11+ and HarmoKnee programmes are useful warm-up protocols for improving concentric hamstring strength in young professional male soccer players. The 11+ programme is more advantageous for its greater concentric hamstring strength improvement compared to the HarmoKnee programme.     

(-)-Amarbellisine, a lycorine-type alkaloid from Amaryllis belladonna L. growing in Egypt

A new lycorine-type alkaloid, named (-)-amarbellisine, was isolated from the bulbs of Egyptian Amaryllis belladonna L. together with the well known alkaloids (-)-lycorine, (-)-pancracine, (+)-vittatine, (+)-11-hydroxyvittatine, and (+)-hippeastrine. The new alkaloid, containing the pyrrolo[de]phenanthridine ring system, was essentially characterised by spectroscopic and optical methods, and proved to be the 2-methoxy-3a,4,5,7,11b,11c-hexahydro-1H-[1,3]dioxolo[4,5-j]pyrrolo[3,2,1-de]phenanthridinol. By using HPTLC technique we also carried out a comparative study of the relative and total alkaloidal content at two different stages of plant growth. Finally, the antimicrobial activity of the isolated alkaloids was assayed.