(+)-Echinobetaine B: isolation, structure elucidation, synthesis and preliminary SAR studies on a new nematocidal betaine from a southern Australian marine sponge, Echinodictyum sp.

The principle nematocidal agent present in a southern Australian marine sponge of the genus Echinodictyum has been isolated and identfied as the novel betaine (+)-echinobetaine B (6), and the structure assigned by spectroscopic analysis has been confirmed by total synthesis. Preliminary SAR conclusions are drawn from analysis of synthetic intermediates and the known marine metabolites zooanemonin (12) and norzooanemonin (13), and the new sponge metabolite norzooanemonin methyl ester (14). The latter compound is reported for the first time from a selection of Australian sponges, including an Axinyssa sp., a Niphates sp., an Axinella sp. and a Ptilocaulis sp.

Glycine receptors: A new therapeutic target in pain pathways

Although glycine receptor Cl- channels (GlyRs) have long been known to mediate inhibitory neurotransmission onto spinal nociceptive neurons, their therapeutic potential for peripheral analgesia has received little attention. However, it has been shown that alpha 3-subunit-containing GlyRs are concentrated into regions of the spinal cord dorsal horn where nociceptive afferents terminate. Furthermore, inflammatory mediators specifically inhibit alpha 3-containing GlyRs, and deletion of the murine alpha 3 gene confers insensitivity to chronic inflammatory pain. This strongly implicates GlyRs in the inflammation-mediated disinhibition of centrally projecting nociceptive neurons. Future therapies aimed at specifically increasing current flux through alpha 3-containing GlyRs may prove effective in providing analgesia.

Molecular determinants of ginkgolide binding in the glycine receptor pore

Ginkgolides are potent blockers of the glycine receptor Cl- channel (GlyR) pore. We sought to identify their binding sites by comparing the effects of ginkgolides A, B and C and bilobalide on alpha 1, alpha 2, alpha 1 beta and alpha 2 beta GlyRs. Bilobalide sensitivity was drastically reduced by incorporation of the beta subunit. In contrast, the sensitivities to ginkgolides B and C were enhanced by beta subunit expression. However, ginkgolide A sensitivity was increased in the alpha 2 beta GlyR relative to the alpha 2 GlyR but not in the alpha 1 beta GlyR relative to the alpha 1 GlyR. We hypothesised that the subunit-specific differences were mediated by residue differences at the second transmembrane domain 2' and 6' pore-lining positions. The increased ginkgolide A sensitivity of the alpha 2 beta GlyR was transferred to the alpha 1 beta GlyR by the G2'A (alpha 1 to alpha 2 subunit) substitution. In addition, the alpha 1 subunit T6'F mutation abolished inhibition by all ginkgolides. As the ginkgolides share closely related structures, their molecular interactions with pore-lining residues were amenable to mutant cycle analysis. This identified an interaction between the variable R2 position of the ginkgolides and the 2' residues of both alpha 1 and beta subunits. These findings provide strong evidence for ginkgolides binding at the 2' pore-lining position.

The dendritic architecture of the cholinergic plexus in the rabbit retina: Selective labeling by glycine accumulation in the presence of sarcosine

The cholinergic amacrine cells in the rabbit retina slowly accumulate glycine to very high levels when the tissue is incubated with excess sarcosine (methylglycine), even though these cells do not normally contain elevated levels of glycine and do not express high-affinity glycine transporters. Because the sarcosine also depletes the endogenous glycine in the glycine-containing amacrine cells and bipolar cells, the cholinergic amacrine cells can be selectively labeled by glycine immunocytochemistry under these conditions. Incubation experiments indicated that the effect of sarcosine on the cholinergic amacrine cells is indirect: sarcosine raises the extracellular concentration of glycine by blocking its re-uptake by the glycinergic amacrine cells, and the excess glycine is probably taken-up by an unidentified low-affinity transporter on the cholinergic amacrine cells. Neurobiotin injection of the On-Off direction-selective (DS) ganglion cells in sarcosine-incubated rabbit retina was combined with glycine immunocytochemistry to examine the dendritic relationships between the DS ganglion cells and the cholinergic amacrine cells. These double-labeled preparations showed that the dendrites of the DS ganglion cells closely follow the fasciculated dendrites of the cholinergic amacrine cells. Each ganglion cell dendrite located within the cholinergic strata is associated with a cholinergic fascicle and, conversely, there are few cholinergic fascicles that do not contain at least one dendrite from an On-Off DS cell. It is not known how the dendritic co-fasciculation develops, but the cholinergic dendritic plexus may provide the initial scaffold, because the dendrites of the On-Off DS cells commonly run along the outside of the cholinergic fascicles. J. Comp. Neurol. 421:1-13, 2000. (C) 2000 Wiley-Liss, Inc.

Histidine acts as a co-germinant with glycine and taurocholate for Clostridium difficile spores

Aims: It is well established that the bile salt sodium taurocholate acts as a germinant for Clostridium difficile spores and the amino acid glycine acts as a co-germinant. The aim of this study was to determine whether any other amino acids act as co-germinants. Methods and Results: Clostridium difficile spore suspensions were exposed to different germinant solutions comprising taurocholate, glycine and an additional amino acid for 1 h before heating shocking (to kill germinating cells) or chilling on ice. Samples were then re-germinated and cultured to recover remaining viable cells. Only five amino acids out of the 19 common amino acids tested (valine, aspartic acid, arginine, histidine and serine) demonstrated co-germination activity with taurocholate and glycine. Of these, only histidine produced high levels of germination (97·9–99·9%) consistently in four strains of Cl. difficile spores. Some variation in the level of germination produced was observed between different PCR ribotypes, and the optimum concentration of amino acids with taurocholate for the germination of Cl. difficile NCTC 11204 spores was 10–100 mmol l-1. Conclusions: Histidine was found to be a co-germinant for Cl. difficile spores when combined with glycine and taurocholate. Significance and Impact of the Study: The findings of this study enhance current knowledge regarding agents required for germination of Cl. difficile spores which may be utilized in the development of novel applications to prevent the spread of Cl. difficile infection.