-
Journal of Clinical Psychopharmacology Aug 2018Supplemental digital content is available in the text.
Supplemental digital content is available in the text.
Topics: Calcium; Digitonin; Humans; Leukocytes, Mononuclear; Lipopolysaccharides; Polymorphism, Single Nucleotide; Pyrazines; Receptor, Serotonin, 5-HT2C; Serotonin
PubMed: 29912796
DOI: 10.1097/JCP.0000000000000914 -
Molecular Therapy. Nucleic Acids Jun 2018Antisense oligonucleotide (AON) therapy for Duchenne muscular dystrophy has drawn great attention in preclinical and clinical trials, but its therapeutic applications...
Antisense oligonucleotide (AON) therapy for Duchenne muscular dystrophy has drawn great attention in preclinical and clinical trials, but its therapeutic applications are still limited due to inefficient delivery. In this study, we investigated a few saponins for their potential to improve delivery performance of an antisense phosphorodiamidate morpholino oligomer (PMO) both in vitro and in vivo. The results showed that these saponins, especially digitonin and tomatine, improve the delivery efficiency of PMO comparable to Endo-Porter-mediated PMO delivery in vitro. The significant enhancement of PMO targeting to dystrophin exon 23 delivery was further observed in mdx mice up to 7-fold with the digitonin as compared to PMO alone. Cytotoxicity of the digitonin and glycyrrhizin was lower than Endo-Porter in vitro and not clearly detected in vivo under the tested concentrations. These results demonstrate that optimization of saponins in molecular size and composition are key factors to achieve enhanced PMO exon-skipping efficiency. The higher efficiency and lower toxicity endow saponins as gene/AON delivery enhancing agents for treating muscular dystrophy or other diseases.
PubMed: 29858054
DOI: 10.1016/j.omtn.2018.02.004 -
Virulence Dec 2018Porphyromonas gingivalis, an opportunistic pathogen usurps gingival epithelial cells (GECs) as primary intracellular niche for its colonization in the oral mucosa....
Porphyromonas gingivalis, an opportunistic pathogen usurps gingival epithelial cells (GECs) as primary intracellular niche for its colonization in the oral mucosa. However, the precise characterization of the intracellular trafficking and fate of P. gingivalis in GECs remains incomplete. Therefore, we employed high-resolution three-dimensional-transmission-electron-microscopy to determine the subcellular location of P. gingivalis in human primary GECs upon invasion. Serial sections of infected-GECs and their tomographic reconstruction depicted ER-rich-double-membrane autophagosomal-vacuoles harboring P. gingivalis. Western-blotting and fluorescence confocal microscopy showed that P. gingivalis significantly induces LC3-lipidation in a time-dependent-manner and co-localizes with LC3, ER-lumen-protein Bip, or ER-tracker, which are major components of the phagophore membrane. Furthermore, GECs that were infected with FMN-green-fluorescent transformant-strain (PgFbFP) and selectively permeabilized by digitonin showed rapidly increasing large numbers of double-membrane-vacuolar-P. gingivalis over 24 hours of infection with a low-ratio of cytosolically free-bacteria. Moreover, inhibition of autophagy using 3-methyladenine or ATG5 siRNA significantly reduced the viability of intracellular P. gingivalis in GECs as determined by an antibiotic-protection-assay. Lysosomal marker, LAMP-1, showed a low-degree colocalization with P. gingivalis (∼20%). PgFbFP was used to investigate the fate of vacuolar- versus cytosolic-P. gingivalis by their association with ubiquitin-binding-adaptor-proteins, NDP52 and p62. Only cytosolic-P. gingivalis had a significant association with both markers, which suggests cytosolically-free bacteria are likely destined to the lysosomal-degradation pathway whereas the vacuolar-P. gingivalis survives. Therefore, the results reveal a novel mechanism for P. gingivalis survival in GECs by harnessing host autophagy machinery to establish a successful replicative niche and persistence in the oral mucosa.
Topics: Autophagosomes; Autophagy; Bacteroidaceae Infections; Endoplasmic Reticulum; Epithelial Cells; Gingiva; Humans; Microbial Viability; Porphyromonas gingivalis
PubMed: 29616874
DOI: 10.1080/21505594.2018.1454171 -
Biochimica Et Biophysica Acta.... Jun 2018The proposal that the respiratory complexes can associate with each other in larger structures named supercomplexes (SC) is generally accepted. In the last decades most...
The proposal that the respiratory complexes can associate with each other in larger structures named supercomplexes (SC) is generally accepted. In the last decades most of the data about this association came from studies in yeasts, mammals and plants, and information is scarce in other lineages. Here we studied the supramolecular association of the FF-ATP synthase (complex V) and the respiratory complexes I, III and IV of the colorless alga Polytomella sp. with an approach that involves solubilization using mild detergents, n-dodecyl-β-D-maltoside (DDM) or digitonin, followed by separation of native protein complexes by electrophoresis (BN-PAGE), after which we identified oligomeric forms of complex V (mainly V and V) and different respiratory supercomplexes (I/IV, I/III, I/IV). In addition, purification/reconstitution of the supercomplexes by anion exchange chromatography was also performed. The data show that these complexes have the ability to strongly associate with each other and form DDM-stable macromolecular structures. The stable V ATPase oligomer was observed by electron-microscopy and the association of the respiratory complexes in the so-called "respirasome" was able to perform in-vitro oxygen consumption.
Topics: Algal Proteins; Detergents; Digitonin; Electron Transport; Electron Transport Complex I; Electron Transport Complex III; Electron Transport Complex IV; Gene Expression; Glucosides; Mitochondria; Oxidative Phosphorylation; Oxygen Consumption; Protein Binding; Volvocida
PubMed: 29540299
DOI: 10.1016/j.bbabio.2018.03.004 -
Virulence Dec 2018The matrix (M) protein of Newcastle disease virus (NDV) is demonstrated to localize in the nucleus via intrinsic nuclear localization signal (NLS), but cellular proteins...
Importin α5 negatively regulates importin β1-mediated nuclear import of Newcastle disease virus matrix protein and viral replication and pathogenicity in chicken fibroblasts.
The matrix (M) protein of Newcastle disease virus (NDV) is demonstrated to localize in the nucleus via intrinsic nuclear localization signal (NLS), but cellular proteins involved in the nuclear import of NDV M protein and the role of M's nuclear localization in the replication and pathogenicity of NDV remain unclear. In this study, importin β1 was screened to interact with NDV M protein by yeast two-hybrid screening. This interaction was subsequently confirmed by co-immunoprecipitation and pull-down assays. In vitro binding studies indicated that the NLS region of M protein and the amino acids 336-433 of importin β1 that belonged to the RanGTP binding region were important for binding. Importantly, a recombinant virus with M/NLS mutation resulted in a pathotype change of NDV and attenuated viral replication and pathogenicity in chicken fibroblasts and SPF chickens. In agreement with the binding data, nuclear import of NDV M protein in digitonin-permeabilized HeLa cells required both importin β1 and RanGTP. Interestingly, importin α5 was verified to interact with M protein through binding importin β1. However, importin β1 or importin α5 depletion by siRNA resulted in different results, which showed the obviously cytoplasmic or nuclear accumulation of M protein and the remarkably decreased or increased replication ability and pathogenicity of NDV in chicken fibroblasts, respectively. Our findings therefore demonstrate for the first time the nuclear import mechanism of NDV M protein and the negative regulation role of importin α5 in importin β1-mediated nuclear import of M protein and the replication and pathogenicity of a paramyxovirus.
Topics: Active Transport, Cell Nucleus; Animals; Cell Line; Chickens; Fibroblasts; Gene Expression Regulation; Immunoprecipitation; Karyopherins; Models, Biological; Newcastle Disease; Newcastle disease virus; Protein Interaction Mapping; Two-Hybrid System Techniques; Viral Matrix Proteins; Virus Replication
PubMed: 29532715
DOI: 10.1080/21505594.2018.1449507 -
Molecules (Basel, Switzerland) Mar 2018Multidrug resistance (MDR) can develop in cancer cells after treatment with anticancer drugs, mainly due to the overexpression of the ATP-binding cassette (ABC)...
BACKGROUND
Multidrug resistance (MDR) can develop in cancer cells after treatment with anticancer drugs, mainly due to the overexpression of the ATP-binding cassette (ABC) transporters. We analyzed the ability of two pungent-tasting alkaloids-capsaicin and piperine from and , respectively-to reverse multidrug resistance in the cancer cell lines Caco-2 and CEM/ADR 5000, which overexpress P-glycoprotein (P-gp) and other ABC transporters.
METHODS
The MTT assay was first used to determine the cytotoxicity of doxorubicin, the alkaloids, and digitonin alone, and then their combinations. Furthermore, rhodamine (Rho) 123 and calcein-AM were used to detect the effects of alkaloids on the activity of P-gp.
RESULTS
Capsaicin and piperine synergistically enhanced the cytotoxicity of doxorubicin in Caco-2 and CEM/ADR 5000 cells. Furthermore, capsaicin and piperine increased the intracellular accumulation of the fluorescent P-glycoprotein (P-gp) substrates rhodamine and calcein and inhibited their efflux from the MDR cell lines.
CONCLUSION
Our study has demonstrated that capsaicin and piperine are P-gp substrates and have potential chemosensitizing activity, which might be interesting for the development of novel modulators of multidrug resistance.
Topics: ATP Binding Cassette Transporter, Subfamily B, Member 1; Alkaloids; Antibiotics, Antineoplastic; Antineoplastic Agents, Phytogenic; Benzodioxoles; Biological Transport; Caco-2 Cells; Capsaicin; Capsicum; Cell Line, Tumor; Doxorubicin; Drug Combinations; Drug Resistance, Multiple; Drug Resistance, Neoplasm; Drug Synergism; Fluoresceins; Gene Expression; HCT116 Cells; Humans; Piper nigrum; Piperidines; Polyunsaturated Alkamides; Rhodamine 123
PubMed: 29498663
DOI: 10.3390/molecules23030557 -
Biochemistry Mar 2018The twin arginine translocation (Tat) system moves folded proteins across the cytoplasmic membrane of bacteria and the thylakoid membrane of plant chloroplasts. Signal...
The twin arginine translocation (Tat) system moves folded proteins across the cytoplasmic membrane of bacteria and the thylakoid membrane of plant chloroplasts. Signal peptide-bearing substrates of the Tat pathway (precursor proteins) are recognized at the membrane by the TatBC receptor complex. The only established preparation of the TatBC complex uses the detergent digitonin, rendering it unsuitable for biophysical analysis. Here we show that the detergent glyco-diosgenin (GDN) can be used in place of digitonin to isolate homogeneous TatBC complexes that bind precursor proteins with physiological specificity. We use this new preparation to quantitatively characterize TatBC-precursor interactions in a fully defined system. Additionally, we show that the GDN-solubilized TatBC complex co-purifies with substantial quantities of phospholipids.
Topics: Chromatography, Reverse-Phase; Detergents; Diosgenin; Escherichia coli; Escherichia coli Proteins; Mass Spectrometry; Membrane Transport Proteins; Native Polyacrylamide Gel Electrophoresis; Surface Plasmon Resonance
PubMed: 29460615
DOI: 10.1021/acs.biochem.8b00026 -
Oxidative Medicine and Cellular... 2017Recently, the discovery of natural compounds capable of modulating nervous system function has revealed new perspectives for a healthier brain. Here, we investigated the...
Recently, the discovery of natural compounds capable of modulating nervous system function has revealed new perspectives for a healthier brain. Here, we investigated the effects of oleic acid (OA) and hydroxytyrosol (HTyr), two important extra virgin olive oil compounds, on lipid synthesis in C6 glioma cells. OA and HTyr inhibited both de novo fatty acid and cholesterol syntheses without affecting cell viability. The inhibitory effect of the individual compounds was more pronounced if OA and HTyr were administered in combination. A reduction of polar lipid biosynthesis was also detected, while triglyceride synthesis was marginally affected. To clarify the lipid-lowering mechanism of these compounds, their effects on the activity of key enzymes of fatty acid biosynthesis (acetyl-CoA carboxylase-ACC and fatty acid synthase-FAS) and cholesterologenesis (3-hydroxy-3-methylglutaryl-CoA reductase-HMGCR) were investigated in situ by using digitonin-permeabilized C6 cells. ACC and HMGCR activities were especially reduced after 4 h of 25 M OA and HTyr treatment. No change in FAS activity was observed. Inhibition of ACC and HMGCR activities is corroborated by the decrease of their mRNA abundance and protein level. Our results indicate a direct and rapid downregulatory effect of the two olive oil compounds on lipid synthesis in C6 cells.
Topics: Animals; Anticholesteremic Agents; Cell Line, Tumor; Cholesterol; Fatty Acid Synthases; Glioma; Lipid Metabolism; Oleic Acid; Olive Oil; Phenylethyl Alcohol; Rats
PubMed: 29435099
DOI: 10.1155/2017/9076052 -
Cell Cycle (Georgetown, Tex.) 2018Human leukemia Jurkat T cells were analyzed for apoptosis and cell cycle by flow cytometry, using the Annexin V/propidium iodide (PI) standard assay, and a simple PI...
Human leukemia Jurkat T cells were analyzed for apoptosis and cell cycle by flow cytometry, using the Annexin V/propidium iodide (PI) standard assay, and a simple PI staining in Triton X-100/digitonin-enriched PI/RNase buffer, respectively. Cells treated with doxorubicin or menadione displayed a very strong correlation between the apoptotic cell fraction measured by the Annexin V/PI assay, and the weight of a secondary cell population that emerged on the forward scatter (FS)/PI plot, as well as on the side scatter (SS)/PI and FL1/PI plots generated from parallel cell cycle recordings. In both cases, the Pearson correlation coefficients were >0.99. In cell cycle determinations, PI fluorescence was detected on FL3 (620/30 nm), and control samples exhibited the expected linear dependence of FL3 on FL1 (525/40 nm) signals. However, increasing doses of doxorubicin or menadione generated a growing subpopulation of cells displaying a definite right-shift on the FS/FL3, SS/FL3 and FL1/FL3 plots, as well as decreased PI fluorescence, indicative of ongoing fragmentation and loss of nuclear DNA. By gating on these events, the resulting fraction of presumably sub-cycling cells (i.e. cells with cleaved DNA, counting sub-G/G, sub-S and sub-G/M cells altogether) was closely similar to the apoptotic rate assessed by Annexin V/PI labeling. Taken together, these findings suggest a possible way to recognize the entire population of cells undergoing apoptotic DNA cleavage and simultaneously determine the cell cycle distribution of non-apoptotic cells in PI-labeled cell samples with various degrees of DNA fragmentation, using a simple and reproducible multiparametric analysis of flow cytometric recordings.
Topics: Antineoplastic Agents; Apoptosis; Cell Cycle; DNA Fragmentation; Doxorubicin; Flow Cytometry; G1 Phase Cell Cycle Checkpoints; G2 Phase Cell Cycle Checkpoints; Humans; Jurkat Cells; Propidium; Vitamin K 3
PubMed: 29417873
DOI: 10.1080/15384101.2018.1426415 -
Traffic (Copenhagen, Denmark) Mar 2018Tail-anchored (TA) proteins insert into their target organelles by incompletely elucidated posttranslational pathways. Some TA proteins spontaneously insert into...
Tail-anchored (TA) proteins insert into their target organelles by incompletely elucidated posttranslational pathways. Some TA proteins spontaneously insert into protein-free liposomes, yet target a specific organelle in vivo. Two spontaneously inserting cytochrome b5 forms, b5-ER and b5-RR, which differ only in the charge of the C-terminal region, target the endoplasmic reticulum (ER) or the mitochondrial outer membrane (MOM), respectively. To bridge the gap between the cell-free and in cellula results, we analyzed targeting in digitonin-permeabilized adherent HeLa cells. In the absence of cytosol, the MOM was the destination of both b5 forms, whereas in cytosol the C-terminal negative charge of b5-ER determined targeting to the ER. Inhibition of the transmembrane recognition complex (TRC) pathway only partially reduced b5 targeting, while strongly affecting the classical TRC substrate synaptobrevin 2 (Syb2). To identify additional pathways, we tested a number of small inhibitors, and found that Eeyarestatin I (ES ) reduced insertion of b5-ER and of another spontaneously inserting TA protein, while not affecting Syb2. The effect was independent from the known targets of ES , Sec61 and p97/VCP. Our results demonstrate that the MOM is the preferred destination of spontaneously inserting TA proteins, regardless of their C-terminal charge, and reveal a novel, substrate-specific ER-targeting pathway.
Topics: Animals; Cell Line; Chlorocebus aethiops; Cytochromes b5; Endoplasmic Reticulum; HeLa Cells; Humans; Mitochondrial Membranes; Protein Domains; Protein Transport; R-SNARE Proteins
PubMed: 29359838
DOI: 10.1111/tra.12550