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The Journal of Biological Chemistry 2021Aggregation of α-synuclein (αS) leads to the hallmark neuropathology of Parkinson's disease (PD) and related synucleinopathies. αS has been described to exist in both...
Aggregation of α-synuclein (αS) leads to the hallmark neuropathology of Parkinson's disease (PD) and related synucleinopathies. αS has been described to exist in both cytosolic and membrane-associated forms, the relative abundance of which has remained unsettled. To study αS under the most relevant conditions by a quantitative method, we cultured and matured rodent primary cortical neurons for >17 days and determined αS cytosol:membrane distribution via centrifugation-free sequential extractions based on the weak ionic detergent digitonin. We noticed that at lower temperatures (4 °C or room temperature), αS was largely membrane-associated. At 37 °C, however, αS solubility was markedly increased. In contrast, the extraction of control proteins (GAPDH, cytosolic; calnexin, membrane) was not affected by temperature. When we compared the relative distribution of the synuclein homologs αS and β-synuclein (βS) under various conditions that differed in temperature and digitonin concentration (200-1200 μg/ml), we consistently found αS to be more membrane-associated than βS. Both proteins, however, exhibited temperature-dependent membrane binding. Under the most relevant conditions (37 °C and 800 μg/ml digitonin, i.e., the lowest digitonin concentration that extracted cytosolic GAPDH to near completion), cytosolic distribution was 49.8% ± 9.0% for αS and 63.6% ± 6.6% for βS. PD-linked αS A30P was found to be largely cytosolic, confirming previous studies that had used different methods. Our work highlights the dynamic nature of cellular synuclein behavior and has important implications for protein-biochemical and cell-biological studies of αS proteostasis, such as testing the effects of genetic and pharmacological manipulations.
Topics: Amino Acid Sequence; Animals; Cell Membrane; Humans; Lentivirus; Neurons; Parkinson Disease; Primary Cell Culture; Protein Aggregates; Protein Aggregation, Pathological; Protein Binding; Rats; Temperature; alpha-Synuclein; beta-Synuclein
PubMed: 33428933
DOI: 10.1016/j.jbc.2021.100271 -
International Journal of Pharmaceutics Nov 2022Migraine is a highly prevalent neurological disease affecting circa 1 billion patients worldwide with severe incapacitating symptoms, which significantly diminishes the...
Migraine is a highly prevalent neurological disease affecting circa 1 billion patients worldwide with severe incapacitating symptoms, which significantly diminishes the quality of life. As self-medication practice, oral administration of triptans is the most common option, despite its relatively slow therapeutic onset and low drug bioavailability. To overcome these issues, here we present, to the best of our knowledge, the first study on the possibility of oral transmucosal delivery of one of the safest triptans, namely eletriptan hydrobromide (EB). Based on a comprehensive set of in vitro and ex vivo experiments, we highlight the conditions required for oral transmucosal delivery, potentially giving rise to similar, or even higher, drug plasma concentrations expected from conventional oral administration. With histology and tissue integrity studies, we conclude that EB neither induces morphological changes nor impairs the integrity of the mucosal barrier following 4 h of exposure. On a cellular level, EB is internalized in human oral keratinocytes within the first 5 min without inducing toxicity at the relevant concentrations for transmucosal delivery. Considering that the pK of EB falls within the physiologically range, we systematically investigated the effect of pH on both solubility and transmucosal permeation. When the pH is increased from 6.8 to 10.4, the drug solubility decreases drastically from 14.7 to 0.07 mg/mL. At pH 6.8, EB gave rise to the highest drug flux and total permeated amount across mucosa, while at pH 10.4 EB shows greater permeability coefficient and thus higher ratio of permeated drug versus applied drug. Permeation experiments with model membranes confirmed the pH dependent permeation profile of EB. The distribution of EB in different cellular compartments of keratinocytes is pH dependent. In brief, high drug ionization leads to higher association with the cell membrane, suggesting ionic interactions between EB and the phospholipid head groups. Moreover, we show that the chemical permeation enhancer DMSO can be used to enhance the drug permeation significantly (i.e., 12 to 36-fold increase). Taken together, this study presents important findings on transmucosal delivery of eletriptan via the oral cavity and paves the way for clinical investigations for a fast and safe migraine treatment.
Topics: Humans; Quality of Life; Dimethyl Sulfoxide; Tryptamines; Administration, Oral; Pharmaceutical Preparations; Migraine Disorders; Phospholipids
PubMed: 36155795
DOI: 10.1016/j.ijpharm.2022.122222 -
Genome Biology Jun 2022The recently developed method TEA-seq and similar DOGMA-seq single cell trimodal omics assays provide unprecedented opportunities for understanding cell biology, but...
The recently developed method TEA-seq and similar DOGMA-seq single cell trimodal omics assays provide unprecedented opportunities for understanding cell biology, but independent evaluation is lacking. We explore the utility of DOGMA-seq compared to the bimodal CITE-seq assay in activated and stimulated human peripheral blood T cells. We find that single cell trimodal omics measurements after digitonin (DIG) permeabilization were generally better than after an alternative "low-loss lysis" (LLL) permeabilization condition. Next, we find that DOGMA-seq with optimized DIG permeabilization and its ATAC library provides more information, although its mRNA and cell surface protein libraries have slightly inferior quality, compared to CITE-seq.
Topics: Benchmarking; Gene Library; High-Throughput Nucleotide Sequencing; Humans; RNA, Messenger; Sequence Analysis, DNA; Single-Cell Analysis
PubMed: 35739535
DOI: 10.1186/s13059-022-02698-8 -
Drug Delivery and Translational Research Sep 2022State-of-the-art in vitro test systems for nanomaterial toxicity assessment are based on dyes and several staining steps which can be affected by nanomaterial...
State-of-the-art in vitro test systems for nanomaterial toxicity assessment are based on dyes and several staining steps which can be affected by nanomaterial interference. Digital holographic microscopy (DHM), an interferometry-based variant of quantitative phase imaging (QPI), facilitates reliable proliferation quantification of native cell populations and the extraction of morphological features in a fast and label- and interference-free manner by biophysical parameters. DHM therefore has been identified as versatile tool for cytotoxicity testing in biomedical nanotechnology. In a comparative study performed at two collaborating laboratories, we investigated the interlaboratory variability and performance of DHM in nanomaterial toxicity testing, utilizing complementary standard operating procedures (SOPs). Two identical custom-built off-axis DHM systems, developed for usage in biomedical laboratories, equipped with stage-top incubation chambers were applied at different locations in Europe. Temporal dry mass development, 12-h dry mass increments and morphology changes of A549 human lung epithelial cell populations upon incubation with two variants of poly(alkyl cyanoacrylate) (PACA) nanoparticles were observed in comparison to digitonin and cell culture medium controls. Digitonin as cytotoxicity control, as well as empty and cabazitaxel-loaded PACA nanocarriers, similarly impacted 12-h dry mass development and increments as well as morphology of A549 cells at both participating laboratories. The obtained DHM data reflected the cytotoxic potential of the tested nanomaterials and are in agreement with corresponding literature on biophysical and chemical assays. Our results confirm DHM as label-free cytotoxicity assay for polymeric nanocarriers as well as the repeatability and reproducibility of the technology. In summary, the evaluated DHM assay could be efficiently implemented at different locations and facilitates interlaboratory in vitro toxicity testing of nanoparticles with prospects for application in regulatory science.
Topics: Digitonin; Holography; Humans; In Vitro Techniques; Microscopy; Reproducibility of Results
PubMed: 35799027
DOI: 10.1007/s13346-022-01207-5 -
Environmental Toxicology and... Aug 2020Diuron, a highly used herbicide worldwide, is metabolized into several toxic metabolites. DCA (3,4-dichloroaniline), DCPU [3-(3, 4-dichlorophenyl)urea] and DCPMU...
Diuron, a highly used herbicide worldwide, is metabolized into several toxic metabolites. DCA (3,4-dichloroaniline), DCPU [3-(3, 4-dichlorophenyl)urea] and DCPMU [3-(3,4-dichlorophenyl)-1-methyl urea] reduced viability of human placental choriocarcinoma BeWo, human breast adenocarcinoma MCF-7 and human colon adenocarcinoma Caco-2 cells as judged by the MTT assay, where color formation is dependent on functional mitochondria in viable cells. Based on the IC values in BeWo cells the order of cytotoxicity was DCA > DCPU > diuron > DCPMU, and in Caco-2 cells DCPMU > DCPU > DCA, diuron. In MCF-7 cells, only DCPU had an IC within the range of the concentrations used. In the PI-digitonin viability assay, only the highest concentration (200 μM) of DCPU caused a statistically significant decrease in viability in any cell line. There was no correlation between cytotoxicity and ROS production. This indicates that diuron metabolites are toxic in cells of human origin with mitochondria as the target, but ROS not the likely mechanism.
Topics: Cell Line, Tumor; Cell Survival; Diuron; Herbicides; Humans; Reactive Oxygen Species
PubMed: 32416162
DOI: 10.1016/j.etap.2020.103409 -
Methods in Molecular Biology (Clifton,... 2024Immunocytochemistry combined with confocal or superresolution microscopy allows us to observe molecular localization and intracellular structures. However, it is...
Immunocytochemistry combined with confocal or superresolution microscopy allows us to observe molecular localization and intracellular structures. However, it is challenging to analyze individual neurons in brain tissue, where neurons are densely packed. In contrast, we can easily observe structures such as the axonal growth cone and dendritic spines in dissociated individual neurons. Thus, the immunocytochemistry of primary cultured neurons is often used because it reflects the in vivo condition at least in part. Here, we describe a method for indirect fluorescence immunocytochemistry of primary cultured neurons from the embryonic cerebral cortex. This involves multiple steps including fixation, permeabilization, and antibody reaction, and in particular, we introduce an optimized protocol for permeabilization to enable the precise localization of target molecules.
Topics: Immunohistochemistry; Antibodies; Cerebral Cortex; Growth Cones; Neurons
PubMed: 38630229
DOI: 10.1007/978-1-0716-3810-1_15 -
Journal of Plant Research Mar 2022The assembly process of photosystem II (PSII) requires several auxiliary proteins to form assembly intermediates. In plants, early assembly intermediates comprise D1 and...
The assembly process of photosystem II (PSII) requires several auxiliary proteins to form assembly intermediates. In plants, early assembly intermediates comprise D1 and D2 subunits of PSII together with a few auxiliary proteins including at least ONE-HELIX PROTEIN1 (OHP1), OHP2, and HIGH-CHLOROPHYLL FLUORESCENCE 244 (HCF244) proteins. Herein, we report the basic characterization of the assembling intermediates, which we purified from Arabidopsis transgenic plants overexpressing a tagged OHP1 protein and named the OHP1 complexes. We analyzed two major forms of OHP1 complexes by mass spectrometry, which revealed that the complexes consist of OHP1, OHP2, and HCF244 in addition to the PSII subunits D1, D2, and cytochrome b. Analysis of chlorophyll fluorescence showed that a major form of the complex binds chlorophyll a and carotenoids and performs quenching with a time constant of 420 ps. To identify the localization of the auxiliary proteins, we solubilized thylakoid membranes using a digitonin derivative, glycodiosgenin, and separated them into three fractions by ultracentrifugation, and detected these proteins in the loose pellet containing the stroma lamellae and the grana margins together with two chlorophyll biosynthesis enzymes. The results indicated that chlorophyll biosynthesis and assembly may take place in the same compartments of thylakoid membranes. Inducible suppression of the OHP2 mRNA substantially decreased the OHP2 protein in mature Arabidopsis leaves without a significant reduction in the maximum quantum yield of PSII under low-light conditions, but it compromised the yields under high-light conditions. This implies that the auxiliary protein is required for acclimation to high-light conditions.
Topics: Arabidopsis; Arabidopsis Proteins; Chlorophyll; Chlorophyll A; Photosystem II Protein Complex; Thylakoids
PubMed: 35146632
DOI: 10.1007/s10265-022-01376-x -
Free Radical Biology & Medicine Dec 2021Permeable cell models have contributed much to the progress in mitochondrial research. Optimization of permeabilization is required to make the cell's plasma membrane...
Permeable cell models have contributed much to the progress in mitochondrial research. Optimization of permeabilization is required to make the cell's plasma membrane permeable to small molecules while keeping the intracellular organelles and their membranes intact and fully functional. Here we report our assessment and optimization of commonly used permeabilizing agents including different saponin preparations, digitonin, and recombinant perfringolysin O employing a new electron flow based mitochondrial assay technology that utilizes a colorimetric redox dye. The results of this study provide guidance in optimizing the conditions for mitochondrial function assays with permeabilized cells using the novel redox dye-based format.
Topics: Cell Membrane; Cell Membrane Permeability; Electrons; Mitochondria; Oxidation-Reduction
PubMed: 34656699
DOI: 10.1016/j.freeradbiomed.2021.10.014 -
Digitonin concentration is determinant for mitochondrial supercomplexes analysis by BlueNative page.Biochimica Et Biophysica Acta.... Jan 2021The BlueNative page (BNGE) gel has been the reference technique for studying the electron transport chain organization since it was established 20 years ago. Although...
The BlueNative page (BNGE) gel has been the reference technique for studying the electron transport chain organization since it was established 20 years ago. Although the migration of supercomplexes has been demonstrated being real, there are still several concerns about its ability to reveal genuine interactions between respiratory complexes. Moreover, the use of different solubilization conditions generates conflicting interpretations. Here, we thoroughly compare the impact of different digitonin concentrations on the liquid dispersions' physical properties and correlate with the respiratory complexes' migration pattern and supercomplexes. Our results demonstrate that digitonin concentration generates liquid dispersions with specific size and variability critical to distinguish between a real association of complexes from being trapped in the same micelle.
Topics: Animals; Digitonin; Electron Transport Complex I; Mice; Mitochondria, Heart; Mitochondria, Liver; Mitochondrial Proteins; Native Polyacrylamide Gel Electrophoresis
PubMed: 33129827
DOI: 10.1016/j.bbabio.2020.148332 -
Forensic Toxicology Jan 2023AMB-FUBINACA is a synthetic cannabinoid receptor agonist (SCRA) which is primarily metabolised by hepatic enzymes producing AMB-FUBINACA carboxylic acid. The...
PURPOSE
AMB-FUBINACA is a synthetic cannabinoid receptor agonist (SCRA) which is primarily metabolised by hepatic enzymes producing AMB-FUBINACA carboxylic acid. The metabolising enzymes associated with this biotransformation remain unknown. This study aimed to determine if AMB-FUBINACA metabolism could be reduced in the presence of carboxylesterase (CES) inhibitors and recreational drugs commonly consumed with it. The affinity and activity of the AMB-FUBINACA acid metabolite at the cannabinoid type-1 receptor (CB) was investigated to determine the activity of the metabolite.
METHODS
The effect of CES1 and CES2 inhibitors, and delta-9-tetrahydrocannabinol (Δ-THC) on AMB-FUBINACA metabolism were determined using both human liver microsomes (HLM) and recombinant carboxylesterases. Radioligand binding and cAMP assays comparing AMB-FUBINACA and AMB-FUBINACA acid were carried out in HEK293 cells expressing human CB.
RESULTS
AMB-FUBINACA was rapidly metabolised by HLM in the presence and absence of NADPH. Additionally, CES1 and CES2 inhibitors both significantly reduced AMB-FUBINACA metabolism. Furthermore, digitonin (100 µM) significantly inhibited CES1-mediated metabolism of AMB-FUBINACA by ~ 56%, while the effects elicited by Δ-THC were not statistically significant. AMB-FUBINACA acid produced only 26% radioligand displacement consistent with low affinity binding. In cAMP assays, the potency of AMB-FUBINACA was ~ 3000-fold greater at CB as compared to the acid metabolite.
CONCLUSIONS
CES1A1 was identified as the main hepatic enzyme responsible for the metabolism of AMB-FUBINACA to its less potent carboxylic acid metabolite. This biotransformation was significantly inhibited by digitonin. Since other xenobiotics may also inhibit similar SCRA metabolic pathways, understanding these interactions may elucidate why some users experience high levels of harm following SCRA use.
Topics: Humans; Cannabinoids; Dronabinol; Digitonin; HEK293 Cells; Cannabinoid Receptor Agonists
PubMed: 36652070
DOI: 10.1007/s11419-022-00649-3