53 research outputs found
A Fungal Metabolite Asperparaline A Strongly and Selectively Blocks Insect Nicotinic Acetylcholine Receptors: The First Report on the Mode of Action
Asperparalines produced by Aspergillus japonicus JV-23 induce
paralysis in silkworm (Bombyx mori) larvae, but the target
underlying insect toxicity remains unknown. In the present study, we have
investigated the actions of asperparaline A on ligand-gated ion channels
expressed in cultured larval brain neurons of the silkworm using patch-clamp
electrophysiology. Bath-application of asperparaline A (10 µM) had no
effect on the membrane current, but when delivered for 1 min prior to
co-application with 10 µM acetylcholine (ACh), it blocked completely the
ACh-induced current that was sensitive to mecamylamine, a nicotinic
acetylcholine receptor (nAChR)-selective antaogonist. In contrast, 10 µM
asperparaline A was ineffective on the γ-aminobutyric acid- and
L-glutamate-induced responses of the Bombyx larval neurons. The
fungal alkaloid showed no-use dependency in blocking the ACh-induced response
with distinct affinity for the peak and slowly-desensitizing current amplitudes
of the response to 10 µM ACh in terms of IC50 values of 20.2
and 39.6 nM, respectively. Asperparaline A (100 nM) reduced the maximum neuron
response to ACh with a minimal shift in EC50, suggesting that the
alkaloid is non-competitive with ACh. In contrast to showing marked blocking
action on the insect nAChRs, it exhibited only a weak blocking action on chicken
α3β4, α4β2 and α7 nAChRs expressed in Xenopus
laevis oocytes, suggesting a high selectivity for insect over
certain vertebrate nAChRs
Selectivity, efficacy and toxicity studies of UCCB01-144, a dimeric neuroprotective PSD-95 inhibitor
Inhibition of postsynaptic density protein-95 (PSD-95) decouples N-methyl-d-aspartate (NMDA) receptor downstream signaling and results in neuroprotection after focal cerebral ischemia. We have previously developed UCCB01-144, a dimeric PSD-95 inhibitor, which binds PSD-95 with high affinity and is neuroprotective in experimental stroke. Here, we investigate the selectivity, efficacy and toxicity of UCCB01-144 and compare with the monomeric drug candidate Tat-NR2B9c. Fluorescence polarization using purified proteins and pull-downs of mouse brain lysates showed that UCCB01-144 potently binds all four PSD-95-like membrane-associated guanylate kinases (MAGUKs). In addition, UCCB01-144 affected NMDA receptor signaling pathways in ischemic brain tissue. UCCB01-144 reduced infarct size in young and aged male mice at various doses when administered 30 min after permanent middle cerebral artery occlusion, but UCCB01-144 was not effective in young male mice when administered 1 h post-ischemia or in female mice. Furthermore, UCCB01-144 was neuroprotective in a transient stroke model in rats, and in contrast to Tat-NR2B9c, high dose of UCCB01-144 did not lead to significant changes in mean arterial blood pressure or heart rate. Overall, UCCB01-144 is a potent MAGUK inhibitor that reduces neurotoxic PSD-95-mediated signaling and improves neuronal survival following focal brain ischemia in rodents under various conditions and without causing cardiovascular side effects, which encourages further studies towards clinical stroke trials
1H NMR-based metabolomics combined with HPLC-PDA-MS-SPE-NMR for investigation of standardized Ginkgo biloba preparations
Commercial preparations of Ginkgo biloba are very complex mixtures prepared from raw leaf extracts by a series of extraction and prepurification steps. The pharmacological activity is attributed to a number of flavonoid glycosides and unique terpene trilactones (TTLs), with largely uncharacterized pharmacological profiles on targets involved in neurological disorders. It is therefore important to complement existing targeted analytical methods for analysis of Ginkgo biloba preparations with alternative technology platforms for their comprehensive and global characterization. In this work, 1H NMR-based metabolomics and hyphenation of high-performance liquid chromatography, photo-diode array detection, mass spectrometry, solid-phase extraction, and nuclear magnetic resonance spectroscopy (HPLC-PDA-MS-SPE-NMR) were used for investigation of 16 commercially available preparations of Ginkgo biloba. The standardized extracts originated from Denmark, Italy, Sweden, and United Kingdom, and the results show that 1H NMR spectra allow simultaneous assessment of the content as well as identity of flavonoid glycosides and TTLs based on a very simple sample-preparation procedure consisting of extraction, evaporation and reconstitution in acetone-d6. Unexpected or unwanted extract constituents were also easily identified in the 1H NMR spectra, which contrasts traditional methods that depend on UV absorption or MS ionizability and usually require availability of reference standards. Automated integration of 1H NMR spectral segments (buckets or bins of 0.02 ppm width) provides relative distribution plots of TTLs based on their H-12 resonances. The present study shows that 1H NMR-based metabolomics is an attractive method for non-selective and comprehensive analysis of Ginkgo extracts
Abstracts from the 20th International Symposium on Signal Transduction at the Blood-Brain Barriers
https://deepblue.lib.umich.edu/bitstream/2027.42/138963/1/12987_2017_Article_71.pd
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