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Morales Joyner posted an update 6 months, 1 week ago
Photoaffinity labeling (PAL) remains one of the most widely utilized methods of determining protein targets of drugs. Although useful, the scope of this technique has been limited to in vitro applications because of the inability of UV light to penetrate whole organisms. Herein, pigment-free Casper zebrafish were employed to allow in vivo PAL. A methamphetamine-related phenethylamine PAL probe, designated here as 2, demonstrated dose-dependent effects on behavior similar to methamphetamine and permitted concentration-dependent labeling of protein binding partners. Click chemistry was used to analyze binding partners via fluoroimaging. Conjugation to a biotin permitted streptavidin pull-down and proteomic analysis to define direct binding partners of the methamphetamine probe. Bioinformatic analysis revealed the probe was chiefly bound to proteins involved in phagocytosis and mitochondrial function. Future applications of this experimental paradigm combining examination of drug-protein binding interactions alongside neurobehavioral readouts via in vivo PAL will significantly enhance our understanding of drug targets, mechanism(s) of action, and toxicity/lethality.The need for improved medications for the treatment of epilepsy and chronic pain is essential. Epileptic patients typically take multiple antiseizure drugs without complete seizure freedom, and chronic pain is not fully managed with current medications. A positive allosteric modulator (PAM) of α2/3-containing GABAA receptors (5-(8-ethynyl-6-(pyridin-2-yl)-4H-benzoimidazolediazepin-3-yl) oxazole or KRM-II-81 (8) is a lead compound in a series of imidazodiazepines. We previously reported that KRM-II-81 produces broad-based anticonvulsant and antinociceptive efficacy in rodent models and provides a wider margin over motoric side effects than that of other GABAA receptor PAMs. DNase I, Bovine pancreas datasheet The present series of experiments was designed to fill key missing gaps in prior preclinical studies assessing whether KRM-II-81 could be further differentiated from nonselective GABAA receptor PAMs using the anticonvulsant diazepam (DZP) as a comparator. In multiple chemical seizure provocation models in mice, KRM-II-81 was either equally or more efficacious than DZP. Most strikingly, KRM-II-81 but not DZP blocked the development of seizure sensitivity to the chemoconvulsants cocaine and pentylenetetrazol in seizure kindling models. These and predecessor data have placed KRM-II-81 into consideration for clinical development requiring the manufacture of kilogram amounts of good manufacturing practice material. We describe here a novel synthetic route amenable to kilogram quantity production. The new biological and chemical data provide key steps forward in the development of KRM-II-81 (8) as an improved treatment option for patients suffering from epilepsy.The inflammatory microenvironment in a lesion is not conducive to the survival of stem cells. Improving the inflammatory microenvironment may be an alternative strategy to enhance the efficacy of stem cells. We evaluated the therapeutic effect and molecular mechanism of mitsugumin53 (MG53) on lipopolysaccharide (LPS)-induced damage in human umbilical cord mesenchymal stem cells (hUC-MSCs) and in C57/BL6 mice. MG53 significantly promoted the proliferation and migration of hUC-MSCs, protected hUC-MSCs against LPS-induced apoptosis and mitochondrial dysfunction, and reversed LPS-induced inflammatory cytokine release. Furthermore, MG53 combined with hUC-MSCs transplantation improved LPS-induced memory impairment and activated neurogenesis by promoting the migration of hUC-MSCs and enhancing βIII-tubulin and doublecortin (DCX) expression. MG53 protein combined with hUC-MSCs improved the M1/M2 phenotype polarization of microglia accompanied by lower inducible nitric oxide synthase (iNOS) expression and higher arginase 1 (ARG1) expression. MG53 significantly suppressed the expression of tumor necrosis factor α (TNF-α), Toll-like receptor 4 (TLR4), nucleotide oligomerization domain-like receptor protein 3 (NLRP3), cleaved-caspase-1, and interleukin (IL)-1β to alleviate LPS-induced neuroinflammation on hUC-MSCs and C57/BL6 mice. In conclusion, our results indicated that MG53 could protect hUC-MSCs against LPS-induced inflammatory damage and facilitate their efficacy in LPS-treated C57/BL6 mice partly by inhibiting the NLRP3/caspase-1/IL-1β axis.Human adult muscle-type acetylcholine receptors incorporating a reconstructed ancestral β-subunit exhibit reduced single-channel conductance when compared to wild-type. The ancestral and wild-type β-subunits differ by 132 amino acids, including substitution of residues that line the lumen of the channel pore, near its narrowest constriction. Here we show that a single historical substitution in this region of the ancestral β-subunit accounts for the difference in conductance. Furthermore, the contribution of the substituted residue to conductance is dependent upon its ancestral or wild-type background, and it can be modulated by a neighboring residue that has also evolved throughout the β-subunit history. Using an expanded molecular phylogeny, we track the order in which these two mutations occurred and then show that the order in which they are installed upon the ancestral, but not the human, background determines their individual contribution to conductance. Our results show how the contribution of amino acids to acetylcholine receptor conductance is contingent upon their evolutionary history and that the order in which substitutions occurred was important for shaping conductance in the modern-day receptor.Oxidative stress is a hallmark of several aging and trauma related neurological disorders, but the precise details of how altered neuronal activity elicits subcellular redox changes have remained difficult to resolve. Current redox sensitive dyes and fluorescent proteins can quantify spatially distinct changes in reactive oxygen species levels, but multicolor probes are needed to accurately analyze compartment-specific redox dynamics in single cells that can be masked by population averaging. We previously engineered genetically encoded red-shifted redox-sensitive fluorescent protein sensors using a Förster resonance energy transfer relay strategy. Here, we developed a second-generation excitation ratiometric sensor called rogRFP2 with improved red emission for quantitative live-cell imaging. Using this sensor to measure activity-dependent redox changes in individual cultured neurons, we observed an anticorrelation in which mitochondrial oxidation was accompanied by a concurrent reduction in the cytosol. This behavior was dependent on the activity of Complex I of the mitochondrial electron transport chain and could be modulated by the presence of cocultured astrocytes.
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