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Molecules (Basel, Switzerland) Jul 2022The rhizomes of are commonly consumed as food and also used as medicine. However, the metabolic profile of has not been fully revealed yet. Recently, we developed a...
The rhizomes of are commonly consumed as food and also used as medicine. However, the metabolic profile of has not been fully revealed yet. Recently, we developed a novel evergreen species of P. sibiricum. The objectives of this study were to compare the metabolic profiles of two types of , i.e., the newly developed evergreen type (Gtype) and a wide-type (Wtype), by using UHPLC-Q-Orbitrap-MS-based untargeted metabolomics approach. A total of 263 and 258 compounds in the positive and negative modes of the mass spectra were tentatively identified. Distinctively different metabolomic profiles of these two types of were also revealed by principal component analysis (PCA) and principal coordinates analysis (PCoA). Furthermore, by using partial least squares discriminant analysis (PLS-DA) modeling, it was found that, as compared with Wtype, Gtype samples had significantly higher content of oxyberberine, proliferin, alpinetin, and grandisin. On the other hand, 15 compounds, including herniarin, kaempferol 7-neohesperidoside, benzyl beta-primeveroside, vanillic acid, biochanin A, neoschaftoside, benzyl gentiobioside, cornuside, hydroxytyrosol-glucuronide, apigenin-pentosyl-glucoside, obacunone, 13-alpha-(21)-epoxyeurycomanone, vulgarin, digitonin, and 3-formylindole, were discovered to have higher abundance in Wtype samples. These distinguishing metabolites suggest the different beneficial health potentials and flavor attributes of the two types of rhizomes.
Topics: Chromatography, High Pressure Liquid; Mass Spectrometry; Metabolomics; Polygonatum; Rhizome
PubMed: 35897876
DOI: 10.3390/molecules27154685 -
Journal of Fluorescence May 2021The varied applications of nanotechnology have paved way for several breakthroughs in the realm of biomedical technology. In this challenging era when illness...
The varied applications of nanotechnology have paved way for several breakthroughs in the realm of biomedical technology. In this challenging era when illness multiplies, timely and accurate disease diagnosis is very important. Thus, well founded novel approaches matter very much in areas like disease diagnosis and monitoring. Nanomedicine has tremendous implications in the given context. An elevated cholesterol concentration in blood is risky and is associated with cardiovascular diseases (CVD). CVD remains the No. 1 global cause of death and hence there is an urge to understand cholesterol level and take preventive measures. Highly fluorescent graphene quantum dots (GQs) are well known for their biocompatibility, non toxicity and aqueous solubility. Here in we report an easy and sensitive non enzymatic based cholesterol detection using digitonin conjugated graphene quantum dots (GDG). Selectivity studies and the cholesterol detection in human blood serum suggests the probe to be reliable and selective for blood cholesterol monitoring. Digitonin conjugated fluorescent graphene quantumdots, an efficient probe for cholesterol sensing.
Topics: Blood Chemical Analysis; Cholesterol; Graphite; Humans; Quantum Dots
PubMed: 33761068
DOI: 10.1007/s10895-021-02712-5 -
Biochimica Et Biophysica Acta.... Oct 2020Saponins are a diverse group of secondary plant metabolites, some of which display hemolytic toxicity due to plasma membrane permeabilization. This feature is employed... (Comparative Study)
Comparative Study
Saponins are a diverse group of secondary plant metabolites, some of which display hemolytic toxicity due to plasma membrane permeabilization. This feature is employed in biological applications for transferring hydrophilic molecules through cell membranes. Widely used commercial saponins include digitonin and saponins from soap tree bark, both of which constitute complex mixtures of little definition. We assessed the permeabilization power of pure saponins towards cellular membranes in an effort to detect novel properties and to improve existing applications. In a respirometric assay, we characterized half-maximal permeabilization of the plasma membrane for different metabolites, of the mitochondrial outer membrane for cytochrome C and the full solubilization of mitochondrial inner membrane protein complexes. Beyond the complete list as repository for the field, we highlight several findings with direct applicability. First, we identified and validated α-chaconine as alternative permeabilization agent in respirometric assays of cultured cells and isolated synaptosomes, superior to digitonin in its tolerability for mitochondria. Second, we identified glycyrrhizic acid to form exceptionally small pores impermeable for adenosine diphosphate. Third, in a concentration dependent manner, tomatine proved to be able to selectively permeabilize the mitochondrial outer, but not inner membrane, allowing for novel states in which to determine cytochrome C oxidase activity. In summary, we provide a list of the permeabilization properties of 18 pure saponins. The identification of two saponins, namely tomatine and chaconine, with direct usability in improved or novel cell biological applications within this small subgroup demonstrates the tremendous potential for further functional screening of pure saponins.
Topics: Animals; Calorimetry; Cell Membrane Permeability; Electron Transport Complex IV; HEK293 Cells; Humans; Metabolism; Mice; Saponins
PubMed: 32598881
DOI: 10.1016/j.bbabio.2020.148251 -
Pathogens (Basel, Switzerland) Oct 2022In this study, we demonstrate that epimastigotes previously grown in LIT medium supplemented with 20 mM galactose and exposed to sub-lethal concentrations of hydrogen...
In this study, we demonstrate that epimastigotes previously grown in LIT medium supplemented with 20 mM galactose and exposed to sub-lethal concentrations of hydrogen peroxide (100 μM) showed two-fold and five-fold viability when compared to epimastigotes grown in LIT medium supplemented with two different glucose concentrations (20 mM and 1.5 mM), respectively. Similar results were obtained when exposing epimastigotes from all treatments to methylene blue 30 μM. Additionally, through differential centrifugation and the selective permeabilization of cellular membranes with digitonin, we found that phosphoglucomutase activity (a key enzyme in galactose metabolism) occurs predominantly within the cytosolic compartment. Furthermore, after partially permeabilizing epimastigotes with digitonin (0.025 mg × mg of protein), intact glycosomes treated with 20 mM galactose released a higher hexose phosphate concentration to the cytosol in the form of glucose-1-phosphate, when compared to intact glycosomes treated with 20 mM glucose, which predominantly released glucose-6-phosphate. These results shine a light on 's galactose metabolism and its interplay with mechanisms that enable resistance to oxidative stress.
PubMed: 36297231
DOI: 10.3390/pathogens11101174 -
Journal of Visualized Experiments : JoVE Sep 2023An erratum was issued for: High-Resolution Respirometry to Assess Mitochondrial Function in Human Spermatozoa. The Protocol and Representative Result sections...
An erratum was issued for: High-Resolution Respirometry to Assess Mitochondrial Function in Human Spermatozoa. The Protocol and Representative Result sections were updated. Step 2.4.12 of the Protocol was updated from: Finally, inject 1 µL of 5 mM antimycin A (2.5 µM final concentration). This is a complex II inhibitor to discriminate between the mitochondrial and residual oxygen consumption (non-mitochondrial respiration). For the analysis of complex I, add 1 µL of 1 mM rotenone (0.5 µM final concentration), an inhibitor of this complex, instead of AA. Measure the oxygen consumption until the signal decreases and stabilizes. to: Finally, inject 1 µL of 5 mM antimycin A (2.5 µM final concentration). This is a complex III inhibitor to discriminate between the mitochondrial and residual oxygen consumption (non-mitochondrial respiration). For the analysis of complex I, add 1 µL of 1 mM rotenone (0.5 µM final concentration), an inhibitor of this complex, instead of AA. Measure the oxygen consumption until the signal decreases and stabilizes. Figure 3 in the Representative Results section was updated from: Figure 3: Determination of the optimal concentration of digitonin for the permeabilization of human sperm cells. The respiration rates were measured at 37 °C in MRM medium with glutamate, malate, and adenosine diphosphate. (A) Representative respiratory trace. The blue line is the O2 concentration, and the red line represents the O2 flow per volume correlated. (B) Mitochondria respiration rate means ± standard error, n = 4. The red arrow represents the optimal concentration. Abbreviation: dig = digitonin. Please click here to view a larger version of this figure. to: Figure 3: Determination of the optimal concentration of digitonin for the permeabilization of human sperm cells. The respiration rates were measured at 37 °C in MRM medium with glutamate, malate, and adenosine diphosphate. (A) Representative respiratory trace. The blue line is the O2 concentration, and the red line represents the O2 flow per volume correlated. (B) Mitochondria respiration rate means ± standard error, n = 4. The red arrow represents the optimal concentration. Abbreviation: dig = digitonin. Please click here to view a larger version of this figure.
PubMed: 37751599
DOI: 10.3791/6574 -
Cell Structure and Function Jun 2024In metazoans, the nuclear envelope (NE) disassembles during the prophase and reassembles around segregated chromatids during the telophase. The process of NE formation...
In metazoans, the nuclear envelope (NE) disassembles during the prophase and reassembles around segregated chromatids during the telophase. The process of NE formation has been extensively studied using live-cell imaging. At the early step of NE reassembly in human cells, specific pattern-like localization of inner nuclear membrane (INM) proteins, connected to the nuclear pore complex (NPC), was observed in the so-called "core" region and "noncore" region on telophase chromosomes, which corresponded to the "pore-free" region and the "pore-rich" region, respectively, in the early G1 interphase nucleus. We refer to these phenomena as NE subdomain formation. To biochemically investigate this process, we aimed to develop an in vitro NE reconstitution system using digitonin-permeabilized semi-intact mitotic human cells coexpressing two INM proteins, emerin and lamin B receptor, which were labeled with fluorescent proteins. The targeting and accumulation of INM proteins to chromosomes before and after anaphase onset in semi-intact cells were observed using time-lapse imaging. Our in vitro NE reconstitution system recapitulated the formation of the NE subdomain, as in living cells, although chromosome segregation and cytokinesis were not observed. This in vitro NE reconstitution required the addition of a mitotic cytosolic fraction supplemented with a cyclin-dependent kinase inhibitor and energy sources. The cytoplasmic soluble factor(s) dependency of INM protein targeting differed among the segregation states of chromosomes. Furthermore, the NE reconstituted on segregated chromosomes exhibited active nucleocytoplasmic transport competency. These results indicate that the chromosome status changes after anaphase onset for recruiting NPC components.Key words: nuclear envelope reassembly, inner nuclear membrane protein, nuclear pore complex, semi-intact cell, in vitro reconstitution.
PubMed: 38839376
DOI: 10.1247/csf.24003 -
Scientific Reports Aug 2020Valosin-containing protein (VCP)/p97/Cdc48 is an AAA + ATPase associated with many ubiquitin-dependent cellular pathways that are central to protein quality control....
Valosin-containing protein (VCP)/p97/Cdc48 is an AAA + ATPase associated with many ubiquitin-dependent cellular pathways that are central to protein quality control. VCP binds various cofactors, which determine pathway selectivity and substrate processing. Here, we used co-immunoprecipitation and mass spectrometry studies coupled to in silico analyses to identify the Leishmania infantum VCP (LiVCP) interactome and to predict molecular interactions between LiVCP and its major cofactors. Our data support a largely conserved VCP protein network in Leishmania including known but also novel interaction partners. Network proteomics analysis confirmed LiVCP-cofactor interactions and provided novel insights into cofactor-specific partners and the diversity of LiVCP complexes, including the well-characterized VCP-UFD1-NPL4 complex. Gene Ontology analysis coupled with digitonin fractionation and immunofluorescence studies support cofactor subcellular compartmentalization with either cytoplasmic or organellar or vacuolar localization. Furthermore, in silico models based on 3D homology modeling and protein-protein docking indicated that the conserved binding modules of LiVCP cofactors, except for NPL4, interact with specific binding sites in the hexameric LiVCP protein, similarly to their eukaryotic orthologs. Altogether, these results allowed us to build the first VCP protein interaction network in parasitic protozoa through the identification of known and novel interacting partners potentially associated with distinct VCP complexes.
Topics: Computer Simulation; Leishmania infantum; Multiprotein Complexes; Protein Structure, Quaternary; Protozoan Proteins; Valosin Containing Protein
PubMed: 32753747
DOI: 10.1038/s41598-020-70010-4 -
Alternative direct-to-amplification cell lysis techniques for forensically relevant non-sperm cells.Journal of Forensic Sciences Nov 2023While efforts have been made to reduce the pervasive backlog of sexual assault evidence collection kits, the actual laboratory process remains very time-consuming due to...
While efforts have been made to reduce the pervasive backlog of sexual assault evidence collection kits, the actual laboratory process remains very time-consuming due to the requirement of a differential lysis step before DNA purification, as well as intricate mixture analysis towards the end of the DNA workflow. Recently, an alternative, direct-to-amplification sperm lysis method (using 1 M NaOH) was identified. However, a direct cell lysis method for non-sperm cells has not been identified yet. Thus, the primary objective of this work was to find an alternative method that is quick, inexpensive, and does not require multiple purification steps for the lysis of non-sperm cells in sexual assault samples. In this study, vaginal swab samples were lysed with the control method, prepGEM™, as well as six alternative reagents: alkaline buffer with 25-200 mM NaOH, high-salt stain extraction buffer, modified radioimmunoprecipitation assay (RIPA) buffer, mammalian protein extraction reagent (M-PER™), digitonin buffer, and urea/thiourea buffer. Quantification using Quantifiler® Trio of vaginal and semen lysates revealed that the alkaline (25 mM NaOH) and M-PER™ methods were efficient for the lysis of vaginal epithelial cells without substantial sperm cell lysis. Following quantification, analysis of STR profiles from vaginal lysates revealed that the M-PER™ method showed promising results across all metrics examined, including the percentage of detected STR alleles, mean peak heights, peak height ratio, and interlocus balance. Thus, this method was recommended as an alternative to the traditional differential lysis method for non-sperm cells given its ability to produce amplification-ready lysates without any DNA purification step.
Topics: Female; Male; Humans; Semen; DNA Fingerprinting; Sodium Hydroxide; Polymerase Chain Reaction; Spermatozoa; Indicators and Reagents; DNA; Microsatellite Repeats
PubMed: 37779342
DOI: 10.1111/1556-4029.15371 -
Cold Spring Harbor Protocols Feb 2020Differential detergent fractionation of cells is a rapid method for extraction of cytoplasmic and nuclear proteins in preparation of an immunoprecipitation. This method...
Differential detergent fractionation of cells is a rapid method for extraction of cytoplasmic and nuclear proteins in preparation of an immunoprecipitation. This method can be applied for use of adherent or suspension cells and can significantly reduce nonspecific background in an immunoprecipitation by separation of cellular compartments into individual fractions. The lysis of cells by differential detergents permits the rapid extraction of proteins from the cytoplasm (digitonin), the cytoplasmic membranes, and organelles (Triton X-100), and nucleoplasm (Tween/DOC), facilitated through the use of distinct extraction buffers. Cytoplasmic and nuclear matrix proteins as well as DNA are left behind during the detergent-based extraction.
Topics: Animals; Cell Fractionation; Cell Membrane; Cell Nucleus; Cells, Cultured; Cytosol; Detergents; Digitonin; Humans; Immunoprecipitation; Membrane Proteins; Nuclear Proteins; Octoxynol; Proteins
PubMed: 32015004
DOI: 10.1101/pdb.prot098582 -
Journal of Pharmaceutical Sciences Jan 2021The fractionation of enough membrane protein from limited samples is challenging for MS-based quantitative targeted absolute proteomics (QTAP) of drug metabolizing...
Differential Detergent Fractionation of Membrane Protein From Small Samples of Hepatocytes and Liver Tissue for Quantitative Proteomic Analysis of Drug Metabolizing Enzymes and Transporters.
The fractionation of enough membrane protein from limited samples is challenging for MS-based quantitative targeted absolute proteomics (QTAP) of drug metabolizing enzymes (DMEs) and transporters. This study evaluated differential detergent fractionation (DDF) of membrane protein from progressively smaller numbers of primary mouse hepatocytes (5 million down to 50,000 cells) and limited liver tissue (25-50 mg) in quantifying select DMEs and transporters by QTAP. Two non-ionic detergents, digitonin and Triton-X-100, were applied in sequence to permeabilize cells and extract membrane proteins. Comparison was made with a membrane protein extraction kit and with homogenization in hypotonic buffer and subsequent differential centrifugation (DC). DDF produced linear membrane protein yields with increasing hepatocyte numbers and better permeabilization evidenced by the higher ratio of cytosolic to membrane protein yields. DDF produced 5-times more membrane protein from liver tissue than DC. The concentration of DMEs and transporters remained consistent in the fractions prepared by DDF from progressively smaller numbers of hepatocytes, but declined in kit fractions. In liver tissue, the concentrations were comparatively higher in DDF versus kit and DC. In conclusion, sequential digitonin and Triton-X-100 fractionation of membrane protein from limited samples is efficient, reproducible and cost-effective for QTAP of DMEs and transporters.
Topics: Animals; Detergents; Hepatocytes; Liver; Membrane Proteins; Mice; Pharmaceutical Preparations; Proteomics
PubMed: 33148403
DOI: 10.1016/j.xphs.2020.10.037