Functional modulation of AMP-activated protein kinase by cereblon

AbstractMutations in cereblon (CRBN), a substrate binding component of the E3 ubiquitin ligase complex, cause a form of mental retardation in humans. However, the cellular proteins that interact with CRBN remain largely unknown. Here, we report that CRBN directly interacts with the α1 subunit of AMP-activated protein kinase (AMPK α1) and inhibits the activation of AMPK activation. The ectopic expression of CRBN reduces phosphorylation of AMPK α1 and, thus, inhibits the enzyme in a nutrient-independent manner. Moreover, AMPK α1 can be potently activated by suppressing endogenous CRBN using CRBN-specific small hairpin RNAs. Thus, CRBN may act as a negative modulator of the AMPK signaling pathway in vivo

Sidedness of carbamazepine accessibility to voltage-gated sodium channels

Abstract Voltage-gated sodium channels are inhibited by many local anesthetics, antiarrhythmics, and antiepileptic drugs. The local anesthetic lidocaine appears to be able to access its binding site in the sodium channel only from the membrane phase or from the internal face of the channel. In contrast, the antiepileptic drug carbamazepine was found to inhibit voltage-gated sodium channels only with external but not internal application, implying a major difference. We investigated this point using both wholecell and inside-out patch recordings from human Nav1.7 channels in a stable cell line. In the whole-cell configuration, carbamazepine inhibited sodium current within seconds when applied externally but had very little no effect when applied internally for up to 15 minutes, confirming previous results. However, carbamazepine inhibited sodium channels effectively and rapidly when applied to the internal face of the membrane using inside-out patch recording. We found that lidocaine also has little or no effect when applied intracellularly in whole-cell recording but blocks effectively and rapidly when applied to the internal surface using inside-out patches. In contrast, the cationic lidocaine derivative QX-314 (N-ethyl-lidocaine) blocks effectively when applied internally with whole-cell dialysis as well as when applied to inside-out patches. We conclude that carbamazepine and lidocaine access the sodium channel in similar ways and hypothesize that their lack of effect with internal dialysis in whole-cell recording reflects rapid exit through membrane near the pipette recording site. This effect likely limits the ability of any compound with significant membrane permeability to be applied intracellularly by whole-cell dialysis. MOL #90472

Sidedness of Carbamazepine Accessibility to Voltage-Gated Sodium Channels

ABSTRACT Voltage-gated sodium channels are inhibited by many local anesthetics, antiarrhythmics, and antiepileptic drugs. The local anesthetic lidocaine appears to be able to access its binding site in the sodium channel only from the membrane phase or from the internal face of the channel. In contrast, the antiepileptic drug carbamazepine was found to inhibit voltage-gated sodium channels only with external, but not internal, application, implying a major difference. We investigated this point using both wholecell and inside-out patch recordings from human Na v 1.7 channels in a stable cell line. In the whole-cell configuration, carbamazepine inhibited sodium current within seconds when applied externally, but had little or no effect when applied internally for up to 15 minutes, confirming previous results. However, carbamazepine inhibited sodium channels effectively and rapidly when applied to the internal face of the membrane using inside-out patch recording. We found that lidocaine also has little or no effect when applied intracellularly in whole-cell recording, but blocks effectively and rapidly when applied to the internal surface using inside-out patches. In contrast, the cationic lidocaine derivative QX-314 (N-ethyl-lidocaine) blocks effectively when applied internally with whole-cell dialysis, as well as when applied to insideout patches. We conclude that carbamazepine and lidocaine access the sodium channel in similar ways and hypothesize that their lack of effect with internal dialysis in whole-cell recording reflects rapid exit through membrane near the pipette recording site. This effect likely limits the ability of any compound with significant membrane permeability to be applied intracellularly by whole-cell dialysis

Synthesis of Cyclic N-Acyl Amidines by [3 + 2] Cycloaddition of N-Silyl Enamines and Activated Acyl Azides

In this study, we describe the synthesis of cyclic N-acyl amidines from readily available N-heteroarenes. The synthetic methodology utilized the versatile N-silyl enamine intermediates from the hydrosilylation of N-heteroarenes for the [3 + 2] cycloaddition reaction step. We evaluated various acyl azides and selected an electronically activated acyl azide, thereby achieving a reasonable yield of cyclic N-acyl amidines. We analyzed the relationship between the reactivity of each step and the electronic nature of substrates using in situ nuclear magnetic resonance spectroscopy. In addition, we demonstrated gram-scale synthesis using the proposed methodology

Investigating the Kinetic Effect on Structural Evolution of Li_<i>x</i>Ni_0.8Co_0.15Al_0.05O₂ Cathode Materials during the Initial Charge/Discharge

In this work, we investigate the structural evolution and reaction kinetics of Li_<i>x</i>Ni_0.8Co_0.15Al_0.05O₂ (NCA) cathode materials induced by the initial charge/discharge as a function of the state of charge (SOC 50 and 90%) and C-rates (0.1–10C), with a combination of high-resolution transmission electron microscopy (HRTEM) imaging, selected area electron diffraction (SAED), and electron energy loss spectroscopy (EELS). During initial charging, the effects of C-rates on the structural modifications of NCA cathode materials are strongly dependent on how much the lithium is extracted from the pristine NCA. The structural modifications become more substantial as the extent of the charge increases, particularly at higher C-rates. In the highly delithiated state (90% SOC), even the particles charged at the same C-rate show significant variations in the degree of the structural modifications. The changes in the crystallographic and electronic structures at the subsurface scales, which were induced by the initial charging to 90% SOC at the rate of 0.1C, are nearly recovered during the initial discharge, except for the NCA discharged at the rate of 10C. To quantify the extent of the irreversible phase transition at the nanoscale, we have utilized HRTEM imaging and scanning transmission electron microscopy (STEM)–EELS line scanning techniques, which enable us to draw complementary results. This comparative analysis provides valuable information that is useful not only for obtaining a complete understanding of the mechanisms by which the degradation is initiated, but also for improving and designing Ni-rich layered cathode materials with better charging and discharging kinetics

Complete Genome Sequence of a Novel Monopartite Mastrevirus, Soybean Geminivirus B, Isolated from Soybean (Glycine max (L.) Merrill)

Soybean is one of the most important crops in Korea. To identify the viruses infecting soybean, we conducted RNA sequencing with samples displaying symptoms of viral disease. A contig displaying sequence similarity to the known Geminivirus was identified. A polymerase chain reaction (PCR) using two different pairs of back-to-back primers and rolling circle amplification (RCA) confirmed the complete genome of a novel virus named soybean geminivirus B (SGVB), consisting of a circular monopartite DNA genome measuring 2616 nucleotides (nt) in length. SGVB contains four open reading frames (ORFs) and three intergenic regions (IRs). IR1 includes a nonanucleotide origin of replication in the stem-loop structure. Phylogenetic and BLAST analyses demonstrated that SGVB could be a novel virus belonging to the genus Mastrevirus in the family Geminiviridae. We generated infectious clones for SGVB by adding a copy of the IR1 region of SGVB, comparing the V-ori in addition to the full-length genome of SGVB. Using the infectious clones, we observed chlorosis and leaf curling with a latent infection in the inoculated Nicotiana benthamiana plants, while none of the inoculated soybean plants showed any visible symptoms of disease. This study provides the complete genome sequence and infectious clones of a novel Mastrevirus referred to as SGVB from soybean in Korea