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Spectrochimica Acta. Part A, Molecular... Oct 2022The concentration of potassium ion is an important indicator for human health, and its abnormality is often accompanied by various diseases. However, most tools...
The concentration of potassium ion is an important indicator for human health, and its abnormality is often accompanied by various diseases. However, most tools currently used to study potassium ion transport are low throughput. Herein, we reported a new K fluorescent nanoprobe CP1-KS with high selectivity and sensitivity to K (fluorescence enhanced factor was up to 9.91 at 20 mM K). The polymeric fluorescent probe CP1-KS was composed of the small-molecular K indicator KS and amphiphilic copolymer CP1. This sensor can be easily and uniformly dispersed in cell culture medium and is suitable for high throughput analysis. To assess the utility of the probe CP1-KS in biological field, this probe was employed as an extracellular fluorescent probe to monitor the efflux of K from cells (E coli, B. Subtilis 168, Hela and MCF-7 cells) under various stimulation including lysozyme, nigericin, digitonin, and ATP. Results demonstrated that CP1-KS is an effective analysis tool for extracellular K concentration. We believe that the nanoprobe has great potential in antibacterial drug screening, K ionophore function, K channel activity, cell membrane permeability analysis or other K related field in the future.
Topics: Biological Assay; Escherichia coli; Fluorescent Dyes; Humans; Ionophores; Ions; Potassium
PubMed: 35653810
DOI: 10.1016/j.saa.2022.121435 -
Plant Methods Mar 2022Blue Native polyacrylamide gel electrophoresis (BN PAGE) followed by denaturing PAGE is a widely used, convenient and time efficient method to separate thylakoid...
BACKGROUND
Blue Native polyacrylamide gel electrophoresis (BN PAGE) followed by denaturing PAGE is a widely used, convenient and time efficient method to separate thylakoid complexes and study their composition, abundance, and interactions. Previous analyses unravelled multiple monomeric and dimeric/oligomeric thylakoid complexes but, in certain cases, the separation of complexes was not proper. Particularly, the resolution of super- and megacomplexes, which provides important information on functional interactions, still remained challenging.
RESULTS
Using a detergent mixture of 1% (w/V) n-dodecyl-β-D-maltoside plus 1% (w/V) digitonin for solubilisation and 4.3-8% gel gradients for separation as methodological improvements in BN PAGE, several large photosystem (PS) I containing bands were detected. According to BN(/BN)/SDS PAGE and mass spectrometry analyses, these PSI bands proved to be PSI-NADH dehydrogenase-like megacomplexes more discernible in maize bundle sheath thylakoids, and PSI complexes with different light-harvesting complex (LHC) complements (PSI-LHCII, PSI-LHCII*) more abundant in mesophyll thylakoids of lincomycin treated maize. For quantitative determination of the complexes and their comparison across taxa and physiological conditions, sample volumes applicable to the gel, correct baseline determination of the densitograms, evaluation methods to resolve complexes running together, calculation of their absolute/relative amounts and distribution among their different forms are proposed.
CONCLUSIONS
Here we report our experience in Blue/Clear-Native polyacrylamide gel electrophoretic separation of thylakoid complexes, their identification, quantitative determination and comparison in different samples. The applied conditions represent a powerful methodology for the analysis of thylakoid mega- and supercomplexes.
PubMed: 35241118
DOI: 10.1186/s13007-022-00858-2 -
Cells Feb 2022Cytotoxicity quantification of nanoparticles is commonly performed by biochemical assays to evaluate their biocompatibility and safety. We explored quantitative phase...
Cytotoxicity quantification of nanoparticles is commonly performed by biochemical assays to evaluate their biocompatibility and safety. We explored quantitative phase imaging (QPI) with digital holographic microscopy (DHM) as a time-resolved in vitro assay to quantify effects caused by three different types of organic nanoparticles in development for medical use. Label-free proliferation quantification of native cell populations facilitates cytotoxicity testing in biomedical nanotechnology. Therefore, DHM quantitative phase images from measurements on nanomaterial and control agent incubated cells were acquired over 24 h, from which the temporal course of the cellular dry mass was calculated within the observed field of view. The impact of LipImage™ 815 lipidots nanoparticles, as well as empty and cabazitaxel-loaded poly(alkyl cyanoacrylate) nanoparticles on the dry mass development of four different cell lines (RAW 264.7, NIH-3T3, NRK-52E, and RLE-6TN), was observed vs. digitonin as cytotoxicity control and cells in culture medium. The acquired QPI data were compared to a colorimetric cell viability assay (WST-8) to explore the use of the DHM assay with standard biochemical analysis methods downstream. Our results show that QPI with DHM is highly suitable to identify harmful or low-toxic nanomaterials. The presented DHM assay can be implemented with commercial microscopes. The capability for imaging of native cells and the compatibility with common 96-well plates allows high-throughput systems and future embedding into existing experimental routines for in vitro cytotoxicity assessment.
Topics: Biological Assay; Cell Line; Holography; Microscopy; Nanoparticles
PubMed: 35203295
DOI: 10.3390/cells11040644 -
Current Research in Microbial Sciences 2022Chagas disease (CD), caused by , occurs in several countries in Latin America and non-endemic countries. Heterogeneity among population has been the Achilles' heel to...
Chagas disease (CD), caused by , occurs in several countries in Latin America and non-endemic countries. Heterogeneity among population has been the Achilles' heel to find a better treatment for CD. In this study, we characterized the biochemical parameters and mitochondrial bioenergetics of epimastigotes differentiated from eight isolates (I1-I8) obtained from Brazilian CD patients. Molecular analysis of parasites DTUs grouped all of them as TcII. The profile of the growth curves in axenic cultures was distinct among them, except for I1 and I3 and I2 and I4. Doubling times, growth rates, cell body length, and resistance to benznidazole were also significantly different among them. All the isolates were more glucose-dependent than other strains adapted to grow in axenic culture. Mitochondrial bioenergetics analysis showed that each isolate behaved differently regarding oxygen consumption rates in non-permeabilized and in digitonin-permeabilized cells in the presence of a complex II-linked substrate. When complex IV-linked respiratory chain substrate was used to provide electrons to the mitochondrial respiratory chain (MRC), similarity among the isolates was higher. Our findings show that TcII epimastigotes derived from patients' trypomastigotes displayed their own characteristics , highlighting the intra-TcII diversity, especially regarding the functionality of mitochondrial respiratory complexes II and IV. Understanding intraspecific biological features help us to move a step further on our comprehension regarding parasite's survival and adaptability offering clues to improve the development of new therapies for CD.
PubMed: 35199071
DOI: 10.1016/j.crmicr.2022.100110 -
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 -
Journal of Virology Mar 2022To gain more information about the nature of virus factories (VFs), we used a recombinant infectious bursal disease virus (IBDV) expressing split-GFP11 tagged to the...
To gain more information about the nature of virus factories (VFs), we used a recombinant infectious bursal disease virus (IBDV) expressing split-GFP11 tagged to the polymerase (VP1) that we have previously shown is a marker for VFs in infected cells expressing GFP1-10. We found that VFs colocalized with 5-ethynyl uridine in the presence of actinomycin, demonstrating they contained newly synthesized viral RNA, and VFs were visible in infected cells that were fixed and permeabilized with digitonin, demonstrating that they were not membrane bound. Fluorescence recovery after photobleaching (FRAP) a region of interest within the VFs occurred rapidly, recovering from approximately 25% to 87% the original intensity over 146 s, and VFs were dissolved by 1,6-hexanediol treatment, demonstrating they showed properties consistent with liquid-liquid phase separation. There was a lower colocalization of the VF GFP signal with the capsid protein VP2 (Manders' coefficient [MC] 0.6), compared to VP3 (MC, 0.9), which prompted us to investigate the VF ultrastructure by transmission electron microscopy (TEM). In infected cells, paracrystalline arrays (PAs) of virions were observed in the cytoplasm, as well as discrete electron dense regions. Using correlative light and electron microscopy (CLEM), we observed that the electron dense regions correlated with the GFP signal of the VFs, which were distinct from the PAs. In summary, VFs contain newly synthesized viral RNA, are not bound by a membrane, show properties consistent with liquid-liquid phase separation, and are distinct from the PAs observed by TEM. Members of the infect birds, fish and insects, and are responsible for diseases of significant economic importance to the poultry industry and aquaculture. Despite their importance, how they replicate in cells remains poorly understood. Here, we show that the virus factories are not membrane bound, demonstrate properties consistent with liquid-liquid phase separation, and are distinct from the paracrystalline arrays of virions observed by transmission electron microscopy, enhancing our fundamental knowledge of virus replication that could be used to develop strategies to control disease, or optimize their therapeutic application.
Topics: Animals; Birnaviridae; Birnaviridae Infections; Cell Line; Chickens; Infectious bursal disease virus; Microscopy, Electron; Poultry Diseases; RNA, Viral; Viral Replication Compartments; Viral Structural Proteins; Virion; Virus Replication
PubMed: 35138130
DOI: 10.1128/jvi.02024-21 -
Methods in Molecular Biology (Clifton,... 2022Autophagy is deregulated in cancer cells and often activated as a cellular stress response to anticancer therapies. Flow cytometry-based assays enable detection and...
Autophagy is deregulated in cancer cells and often activated as a cellular stress response to anticancer therapies. Flow cytometry-based assays enable detection and quantification of various cellular markers in live or fixed cells. Here, a flow cytometry-based assay to characterize autophagy across the cell cycle is described. This method is based on selective plasma membrane permeabilization with digitonin and extraction of membrane-unbound LC3 protein followed by staining of the autophagosome-bound LC3 protein with antibody and labeling of DNA with propidium iodide. Staining with the LC3 antibody described here can be also combined with the staining of other cellular markers, allowing to quantitatively assess autophagy in relation to different cellular processes by flow cytometry.
Topics: Autophagosomes; Autophagy; Cell Cycle; Flow Cytometry; Microtubule-Associated Proteins
PubMed: 34972986
DOI: 10.1007/978-1-0716-2071-7_5 -
Frontiers in Physiology 2021In response to various pathological stimuli, such as oxidative and energy stress accompanied by high Ca, mitochondria undergo permeability transition (PT) leading to the...
In response to various pathological stimuli, such as oxidative and energy stress accompanied by high Ca, mitochondria undergo permeability transition (PT) leading to the opening of the non-selective PT pores (PTP) in the inner mitochondrial membrane. Opening of the pores at high conductance allows the passage of ions and solutes <1.5 kD across the membrane, that increases colloid osmotic pressure in the matrix leading to excessive mitochondrial swelling. Calcium retention capacity (CRC) reflects maximum Ca overload of mitochondria that occurs just before PTP opening. Quantification of CRC is important for elucidating the effects of different pathological stimuli and the efficacy of pharmacological agents on the mitochondria. Here, we performed a comparative analysis of CRC in mitochondria isolated from H9c2 cardioblasts, and in permeabilized H9c2 cells to highlight the strengths and weaknesses of the CRC technique in isolated cell mitochondria vs. permeabilized cells. The cells were permeabilized by digitonin or saponin, and the Ca-sensitive fluorescence probe Calcium Green-5N was used in both preparations. Results demonstrated the interference of dye-associated fluorescence signals with saponin and the adverse effects of digitonin on mitochondria at high concentrations. Analysis of the CRC in permeabilized cells revealed a higher CRC in the saponin-permeabilized cells in comparison with the digitonin-permeabilized cells. In addition, the mitochondrial CRC in saponin-permeabilized cells was higher than in isolated mitochondria. Altogether, these data demonstrate that the quantification of the mitochondrial CRC in cultured cells permeabilized by saponin has more advantages compared to the isolated mitochondria.
PubMed: 34950052
DOI: 10.3389/fphys.2021.773839 -
International Journal of Biological... Jan 2022Previously we have shown that lactoferrin (LTF), a protein of secondary neutrophilic granules, can be efficiently modified by hypohalous acids (HOCl and HOBr), which are...
Previously we have shown that lactoferrin (LTF), a protein of secondary neutrophilic granules, can be efficiently modified by hypohalous acids (HOCl and HOBr), which are produced at high concentrations during inflammation and oxidative/halogenative stress by myeloperoxidase, an enzyme of azurophilic neutrophilic granules. Here we compared the effects of recombinant human lactoferrin (rhLTF) and its halogenated derivatives (rhLTF-Cl and rhLTF-Br) on functional responses of neutrophils. Our results demonstrated that after halogenative modification, rhLTF lost its ability to induce mobilization of intracellular calcium, actin cytoskeleton reorganization, and morphological changes in human neutrophils. Moreover, both forms of the halogenated rhLTF prevented binding of N-acetylglucosamine-specific plant lectin Triticum vulgaris agglutinin (WGA) to neutrophils and, in contrast to native rhLTF, inhibited respiratory burst of neutrophils induced by N-formyl-L-methionyl-L-leucyl-L-phenylalanine and by two plant lectins (WGA and PHA-L). However, we observed no differences between the effects of rhLTF, rhLTF-Cl, and rhLTF-Br on respiratory burst of neutrophils induced by phorbol 12-myristate 13-acetate (PMA), digitonin, and number of plant lectins with different glycan-binding specificity. Furthermore, all rhLTF forms interfered with PMA- and ionomycin-induced formation of neutrophil extracellular traps. Thus, halogenative modification of LTF is one of the mechanisms involved in modulating a variety of signaling pathways in neutrophils to control their pro-inflammatory activity.
Topics: Acetylglucosamine; Actin Cytoskeleton; Bromates; Calcium; Digitonin; Humans; Hypochlorous Acid; Ionomycin; Lactoferrin; Neutrophils; Recombinant Proteins; Tetradecanoylphorbol Acetate; Triticum; Wheat Germ Agglutinins
PubMed: 34863835
DOI: 10.1016/j.ijbiomac.2021.11.165 -
Molecules (Basel, Switzerland) Oct 2021Saponins, a diverse group of natural compounds, offer an interesting pool of derivatives with biomedical application. In this study, three structurally related...
Spirostanol Saponins from Flowers of and Related Compounds Indicating Cytotoxic Activity and Affecting Nitric Oxide Production Inhibitory Effect in Peritoneal Macrophages.
Saponins, a diverse group of natural compounds, offer an interesting pool of derivatives with biomedical application. In this study, three structurally related spirostanol saponins were isolated and identified from the leek flowers of L. (garden leek). Two of them were identical with the already known leek plant constituents: aginoside (1) and 6-deoxyaginoside (2). The third one was identified as new component of ; however, it was found identical with yayoisaponin A (3) obtained earlier from a mutant of elephant garlic L. It is a derivative of the aginoside (1) with additional glucose in its glycosidic chain, identified by MS and NMR analysis as (2α, 3β, 6β, 25)-2,6-dihydroxyspirostan-3-yl β-D-glucopyranosyl-(1 → 3)-β-D-glucopranosyl-(1 → 2)-[β-D-xylopyranosyl-(1 → 3)]-β-D-glucopyranosyl]-(1 → 4)-β-D-galactopyranoside, previously reported also under the name alliporin. The leek native saponins were tested together with other known and structurally related saponins (tomatonin and digitonin) and with their related aglycones (agigenin and diosgenin) for in vitro cytotoxicity and for effects on NO production in mouse peritoneal cells. The highest inhibitory effects were exhibited by 6-deoxyaginoside. The obtained toxicity data, however, closely correlated with the suppression of NO production. Therefore, an unambiguous linking of obtained bioactivities of saponins with their expected immunobiological properties remained uncertain.
Topics: Allium; Animals; Cell Line; Cell Survival; Flowers; Lipopolysaccharides; Macrophages, Peritoneal; Mice; Mice, Inbred C57BL; Molecular Conformation; Nitric Oxide; Saponins; Spirostans
PubMed: 34770942
DOI: 10.3390/molecules26216533