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Journal of Experimental Botany Feb 2019Microspore embryogenesis is an experimental morphogenic pathway with important applications in basic research and applied plant breeding, but its genetic, cellular, and...
Microspore embryogenesis is an experimental morphogenic pathway with important applications in basic research and applied plant breeding, but its genetic, cellular, and molecular bases are poorly understood. We applied a multidisciplinary approach using confocal and electron microscopy, detection of Ca2+, callose, and cellulose, treatments with caffeine, digitonin, and endosidin7, morphometry, qPCR, osmometry, and viability assays in order to study the dynamics of cell wall formation during embryogenesis induction in a high-response rapeseed (Brassica napus) line and two recalcitrant rapeseed and eggplant (Solanum melongena) lines. Formation of a callose-rich subintinal layer (SL) was common to microspore embryogenesis in the different genotypes. However, this process was directly related to embryogenic response, being greater in high-response genotypes. A link could be established between Ca2+ influx, abnormal callose/cellulose deposition, and the genotype-specific embryogenic competence. Callose deposition in inner walls and SLs are independent processes, regulated by different callose synthases. Viability and control of internal osmolality are also related to SL formation. In summary, we identified one of the causes of recalcitrance to embryogenesis induction: a reduced or absent protective SL. In responding genotypes, SLs are markers for changes in cell fate and serve as osmoprotective barriers to increase viability in imbalanced in vitro environments. Genotype-specific differences relate to different responses against abiotic (heat/osmotic) stresses.
Topics: Brassica napus; Cell Differentiation; Genotype; Pollen; Seeds; Solanum melongena
PubMed: 30715473
DOI: 10.1093/jxb/ery458 -
The Journal of General and Applied... Sep 2019After being translocated into the ER lumen, membrane and secretory proteins are transported from the ER to the early Golgi by COPII vesicles. Incorporation of these...
After being translocated into the ER lumen, membrane and secretory proteins are transported from the ER to the early Golgi by COPII vesicles. Incorporation of these cargo proteins into COPII vesicles are facilitated either by direct interaction of cargo proteins with COPII coat proteins or by ER exit adaptor proteins which mediate the interaction of cargo proteins with COPII coat proteins. Svp26 is one of the ER exit adaptor proteins in yeast Saccharomyces cerevisiae. ER exit of several type II membrane proteins have been reported to be facilitated by Svp26. We demonstrate here that efficient incorporation of Mnt2 and Mnt3 into COPII vesicles is also dependent on the function of Svp26. Mnt2 and Mnt3 are Golgi-localized α-1,3-mannosyltransferases with type II membrane topology involved in protein O-glycosylation. Immunoisolation of the yeast Golgi subcompartments quantitatively showed that Mnt2 and Mnt3 are more abundant in the early Golgi fraction than in the late Golgi fraction. Subcellular fractionation and fluorescence microscopy showed that deletion of the SVP26 gene results in the accumulation of Mnt2 and Mnt3 in ER. Using an in vitro COPII vesicle formation assay, we further demonstrate that Svp26 facilitates incorporation of Mnt2 and Mnt3 into COPII vesicles. Finally, we showed that Mnt2 and Mnt3 were co-immunoprecipitated with Svp26 from digitonin-solubilized membranes. These results indicate that Svp26 functions as an ER exit adaptor protein of Mnt2 and Mnt3.
Topics: Biological Transport; Endoplasmic Reticulum; Golgi Apparatus; Mannosyltransferases; Protein Binding; Saccharomyces cerevisiae; Saccharomyces cerevisiae Proteins; Vesicular Transport Proteins
PubMed: 30700649
DOI: 10.2323/jgam.2018.09.001 -
Current Protocols in Cytometry Apr 2019Flow cytometry approaches combined with a genetically encoded targeted fluorescent biosensor are used to determine the subcellular compartmental availability of the...
Flow cytometry approaches combined with a genetically encoded targeted fluorescent biosensor are used to determine the subcellular compartmental availability of the oxidized form of nicotinamide adenine dinucleotide (NAD ). The availability of free NAD can affect the activities of NAD -consuming enzymes such as sirtuin, PARP/ARTD, and cyclic ADPR-hydrolase family members. Many methods for measuring the NAD available to these enzymes are limited because they cannot determine free NAD as it exists in various subcellular compartments distinctly from bound NAD or NADH. Here, an approach to express the sensor in mammalian cells, monitor NAD -dependent fluorescence intensity changes using flow cytometry approaches, and analyze data obtained is described. The benefit of flow cytometry approaches with the NAD sensor is the ability to monitor compartmentalized free NAD fluctuations simultaneously within many cells, which greatly facilitates analyses and calibration. © 2018 by John Wiley & Sons, Inc.
Topics: Acrylamides; Biosensing Techniques; Calibration; Digitonin; Enzyme Inhibitors; Flow Cytometry; Fluorescence; HeLa Cells; Humans; Hydrogen-Ion Concentration; Intracellular Space; Mitochondria; NAD; Nicotinamide Phosphoribosyltransferase; Piperidines; Small Molecule Libraries; Statistics as Topic
PubMed: 30556645
DOI: 10.1002/cpcy.54 -
Scientific Reports Nov 2018This study sought to elucidate how oligomycin, an ATP synthase blocker, leads to underestimation of maximal oxygen consumption rate (OCR) and spare respiratory capacity...
This study sought to elucidate how oligomycin, an ATP synthase blocker, leads to underestimation of maximal oxygen consumption rate (OCR) and spare respiratory capacity (SRC) in tumor cells. T98G and U-87MG glioma cells were titrated with the protonophore CCCP to induce OCR. The presence of oligomycin (0.3-3.0 µg/mL) led to underestimation of OCR and a consequent decrease in SRC values of between 25% and 40% in medium containing 5.5 or 11 mM glucose. The inhibitory effect of oligomycin on CCCP-induced OCR did not occur when glutamine was the metabolic substrate or when the glycolytic inhibitor 2-deoxyglucose was present. ATP levels were reduced and ADP/ATP ratios increased in cells treated with CCCP, but these changes were minimized when oligomycin was used to inhibit reverse activity of ATP synthase. Exposing digitonin-permeabilized cells to exogenous ATP, but not ADP, resulted in partial inhibition of CCCP-induced OCR. We conclude that underestimation of OCR and SRC in tumor cells when ATP synthase is inhibited is associated with high glycolytic activity and that the glycolytic ATP yield may have an inhibitory effect on the metabolism of respiratory substrates and cytochrome c oxidase activity. Under CCCP-induced OCR, oligomycin preserves intracellular ATP by inhibiting ATP synthase reverse activity.
Topics: Adenosine Triphosphate; Cell Line, Tumor; Deoxyglucose; Electron Transport; Glycolysis; Humans; Mitochondria; Mitochondrial Proton-Translocating ATPases; Neoplasms; Oligomycins; Oxygen Consumption
PubMed: 30478338
DOI: 10.1038/s41598-018-35679-8 -
Saponins enhance exon skipping of 2'-O-methyl phosphorothioate oligonucleotide in vitro and in vivo.Drug Design, Development and Therapy 2018Antisense oligonucleotide (ASO)-mediated exon skipping has been feasible and promising approach for treating Duchenne muscular dystrophy (DMD) in preclinical and...
BACKGROUND
Antisense oligonucleotide (ASO)-mediated exon skipping has been feasible and promising approach for treating Duchenne muscular dystrophy (DMD) in preclinical and clinical trials, but its therapeutic applications remain challenges due to inefficient delivery.
METHODS
We investigated a few Saponins for their potential to improve delivery performance of an antisense 2'-Omethyl phosphorothioate RNA (2'-OMePS) in muscle cells and in dystrophic mice. This study was carried out by evaluating these Saponins' toxicity, cellular uptake, transduction efficiency in vitro, and local delivery in vivo for 2'-OMePS, as well as affinity study between Saponin and 2'-OMePS.
RESULTS
The results showed that these Saponins, especially Digitonin and Tomatine, enhance the delivery of 2'-OMePS with comparable efficiency to Lipofectamine 2k (LF-2k) -mediated delivery in vitro. Significant performance was further observed in mice, up to 10-fold with the Digitonin as compared to 2'-OMePS alone. Cytotoxicity of the Digitonin and Glycyrrhizin was much lower than LF-2k and not clearly detected in vivo under the tested concentrations.
CONCLUSION
This study potentiates Saponins as delivery vehicle for 2'-OMePS in vivo for treating DMD or other diseases.
Topics: Animals; Cell Line; Cell Survival; Dose-Response Relationship, Drug; Drug Carriers; Drug Delivery Systems; Exons; Lipids; Mice; Mice, Inbred C57BL; Molecular Conformation; Oligonucleotides, Antisense; Phosphorothioate Oligonucleotides; Saponins; Structure-Activity Relationship
PubMed: 30464402
DOI: 10.2147/DDDT.S179008 -
Medicines (Basel, Switzerland) Nov 2018We studied the effect of three plant extracts (, , ) and six of their major secondary metabolites (glycyrrhizic acid, 18β glycyrrhetinic acid, liquiritigenin,...
We studied the effect of three plant extracts (, , ) and six of their major secondary metabolites (glycyrrhizic acid, 18β glycyrrhetinic acid, liquiritigenin, isoliquiritigenin, paeoniflorin, ursolic acid) on the multidrug resistant human colon cancer cell line Caco-2 and human leukemia cell line CEM/ADR 5000 as compared to the corresponding sensitive cell line CCRF-CEM, and human colon cancer cells HCT-116, which do not over-express ATP-binding cassette (ABC) transporters. The cytotoxicity of single substances in sensitive and resistant cells was investigated by MTT assay. We also applied combinations of extracts or single compounds with the chemotherapeutic agent doxorubicin or doxorubicin plus the saponin digitonin. The intracellular retention of the ABC transporter substrates rhodamine 123 and calcein was examined by flow cytometry to explore the effect of the substances on the activity of ABC transporters P-glycoprotein and MRP1. Real-time PCR was applied to analyse the gene expression changes of ABCB1, ABCC1, caspase 3, caspase 8, AhR, CYP1A1, and GSTP1 in resistant cells under the treatment of the substances. All the substances moderately inhibited cell growth in sensitive and resistant cells to some degree. Whereas ursolic acid showed IC of 14 and 22 µM in CEM/ADR 5000 and Caco-2 cells, respectively, glycyrrhizic acid and paeoniflorin were inactive with IC values above 400 μM. Except for liquiritigenin and isoliquiritigenin, all the other substances reversed MDR in CEM/ADR 5000 and Caco-2 cells to doxorubicin. Ue, ga, 18ga, and urs were powerful reversal agents. In CEM/ADR 5000 cells, high concentrations of all the substances, except extract, increased calcein or rhodamine 123 retention in a dose-dependent manner. In Caco-2 cells, all the substances, except liquiritigenin, retained rhodamine 123 in a dose-dependent manner. We also examined the effect of the plant secondary metabolite (PSM) panel on the expression of ABCB1, ABCC1, caspase 3, caspase 8, AhR, CYP1A1, and GSTP1 genes in MDR cells. The extracts and individual PSM could reverse MDR in CEM/ADR 5000 and Caco-2 cells, which overexpress ABC transporters, in two- and three-drug combinations. Most of the PSM also inhibited the activity of ABC transporters to some degree, albeit at high concentrations. Ue, ga, 18ga, and urs were identified as potential multidrug resistance (MDR) modulator candidates, which need to be characterized and validated in further studies.
PubMed: 30428619
DOI: 10.3390/medicines5040123 -
PloS One 2018The calcium binding protein ALG-2 is upregulated in several types of cancerous tissues and cancer cell death may be a consequence of ALG-2 downregulation. Novel research...
The calcium binding protein ALG-2 is upregulated in several types of cancerous tissues and cancer cell death may be a consequence of ALG-2 downregulation. Novel research suggests that ALG-2 is involved in membrane repair mechanisms, in line with several published studies linking ALG-2 to processes of membrane remodeling and transport, which may contribute to the fitness of cells or protect them from damage. To investigate the involvement of ALG-2 in cell recovery after membrane damage we disrupted the PDCD6 gene encoding the ALG-2 protein in DT-40 cells and exposed them to electroporation. ALG-2 knock-out cells were more sensitive to electroporation as compared to wild type cells. This phenotype could be reversed by reestablishing ALG-2 expression confirming that ALG-2 plays an important role in cell recovery after plasma membrane damage. We found that overexpression of wild type ALG-2 but not a mutated form unable to bind Ca2+ partially protected HeLa cells from digitonin-induced cell death. Further, we were able to inhibit the cell protective function of ALG-2 after digitonin treatment by adding a peptide with the ALG-2 binding sequence of ALIX, which has been proposed to serve as the ALG-2 downstream target in a number of processes including cell membrane repair. Our results suggest that ALG-2 may serve as a novel therapeutic target in combination with membrane damaging interventions.
Topics: Animals; Apoptosis Regulatory Proteins; Avian Proteins; Calcium; Calcium-Binding Proteins; Cations, Divalent; Cell Line; Cell Membrane; Cell Survival; Chickens; Digitonin; Electroporation; Gene Knockout Techniques; HeLa Cells; Humans; Mutation
PubMed: 30240438
DOI: 10.1371/journal.pone.0204520 -
PloS One 2018A prominent theory of cell death in myocardial ischemia/reperfusion (I/R) posits that the primary and pivotal step of irreversible cell injury is the opening of the...
A prominent theory of cell death in myocardial ischemia/reperfusion (I/R) posits that the primary and pivotal step of irreversible cell injury is the opening of the mitochondrial permeability transition (MPT) pore. However, the predominantly positive evidence of protection against infarct afforded by the MPT inhibitor, Cyclosporine A (CsA), in experimental studies is in stark contrast with the overall lack of benefit found in clinical trials of CsA. One reason for the discrepancy might be the fact that relatively short experimental ischemic episodes (<1 hour) do not represent clinically-realistic durations, usually exceeding one hour. Here we tested the hypothesis that MPT is not the primary event of cell death after prolonged (60-80 min) episodes of global ischemia. We used confocal microcopy in Langendorff-perfused rabbit hearts treated with the electromechanical uncoupler, 2,3-Butanedione monoxime (BDM, 20 mM) to allow tracking of MPT and sarcolemmal permeabilization (SP) in individual ventricular myocytes. The time of the steepest drop in fluorescence of mitochondrial membrane potential (ΔΨm)-sensitive dye, TMRM, was used as the time of MPT (TMPT). The time of 20% uptake of the normally cell-impermeable dye, YO-PRO1, was used as the time of SP (TSP). We found that during reperfusion MPT and SP were tightly coupled, with MPT trending slightly ahead of SP (TSP-TMPT = 0.76±1.31 min; p = 0.07). These coupled MPT/SP events occurred in discrete myocytes without crossing cell boundaries. CsA (0.2 μM) did not reduce the infarct size, but separated SP and MPT events, such that detectable SP was significantly ahead of MPT (TSP -TMPT = -1.75±1.28 min, p = 0.006). Mild permeabilization of cells with digitonin (2.5-20 μM) caused coupled MPT/SP events which occurred in discrete myocytes similar to those observed in Control and CsA groups. In contrast, deliberate induction of MPT by titration with H2O2 (200-800 μM), caused propagating waves of MPT which crossed cell boundaries and were uncoupled from SP. Taken together, these findings suggest that after prolonged episodes of ischemia, SP is the primary step in myocyte death, of which MPT is an immediate and unavoidable consequence.
Topics: Animals; Cardiotonic Agents; Cell Death; Cell Membrane Permeability; Cyclosporine; Female; Male; Microscopy, Confocal; Myocardial Ischemia; Myocardial Reperfusion Injury; Rabbits; Sarcolemma
PubMed: 29975744
DOI: 10.1371/journal.pone.0200301 -
Bio-protocol Jul 2018The hierarchical composition and interactions of the labile thylakoid protein complexes can be assessed by sequential 2D-native gel-electrophoresis system. Mild...
The hierarchical composition and interactions of the labile thylakoid protein complexes can be assessed by sequential 2D-native gel-electrophoresis system. Mild non-ionic detergent digitonin is used to solubilize labile protein super-and megacomplexes, which are then separated with first-dimension blue native polyacrylamide gel electrophoresis (1D-BN-PAGE). The digitonin derived protein complexes are further solubilized with stronger detergent, β-DM, and subsequently separated on an orthogonal 2D-BN-PAGE to release smaller protein subcomplexes from the higher-order supercomplexes. Here we describe a detailed method for 2D-BN-PAGE analysis of thylakoid protein complexes from .
PubMed: 34395737
DOI: 10.21769/BioProtoc.2899 -
Biophysical Journal Jun 2018Pulsed electric fields with microsecond pulse width (μsPEFs) are used clinically; namely, irreversible electroporation/Nanoknife is used for soft tissue tumor ablation....
Pulsed electric fields with microsecond pulse width (μsPEFs) are used clinically; namely, irreversible electroporation/Nanoknife is used for soft tissue tumor ablation. The μsPEF pulse parameters used in irreversible electroporation (0.5-1 kV/cm, 80-100 pulses, ∼100 μs each, 1 Hz frequency) may cause an internal field to develop within the cell because of the disruption of the outer cell membrane and subsequent penetration of the electric field. An internal field may disrupt voltage-sensitive mitochondria, although the research literature has been relatively unclear regarding whether such disruptions occur with μsPEFs. This investigation reports the influence of clinically used μsPEF parameters on mitochondrial respiration in live cells. Using a high-throughput Agilent Seahorse machine, it was observed that μsPEF exposure comprising 80 pulses with amplitudes of 600 or 700 V/cm did not alter mitochondrial respiration in 4T1 cells measured after overnight postexposure recovery. To record alterations in mitochondrial function immediately after μsPEF exposure, high-resolution respirometry was used to measure the electron transport chain state via responses to glutamate-malate and ADP and mitochondrial membrane potential via response to carbonyl cyanide-p-trifluoromethoxyphenylhydrazone. In addition to measuring immediate mitochondrial responses to μsPEF exposure, measurements were also made on cells permeabilized using digitonin and those with compromised cytoskeleton due to actin depolymerization via treatment with the drug latrunculin B. The former treatment was used as a control to tease out the effects of plasma membrane permeabilization, whereas the latter was used to investigate indirect effects on the mitochondria that may occur if μsPEFs impact the cytoskeleton on which the mitochondria are anchored. Based on the results, it was concluded that within the pulse parameters tested, μsPEFs alone do not hinder mitochondrial physiology but can be used to impact the mitochondria upon compromising the actin. Mitochondrial susceptibility to μsPEF after actin depolymerization provides, to our knowledge, a novel avenue for cancer therapeutics.
Topics: Actin Cytoskeleton; Animals; Cell Line, Tumor; Cell Respiration; Cytoskeleton; Electricity; Membrane Potential, Mitochondrial; Mice; Mitochondria; Permeability
PubMed: 29925031
DOI: 10.1016/j.bpj.2018.04.047