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MethodsX 2019Key mitochondrial processes are known to be widely conserved throughout the eukaryotic domain. However, the scarce availability of working materials may restrict the...
Key mitochondrial processes are known to be widely conserved throughout the eukaryotic domain. However, the scarce availability of working materials may restrict the assessment of such mitochondrial activities in several working models. Pollen tube mitochondrial studies represent one example of this, where tests have been often restricted due the physical impossibility of performing experiments with isolated mitochondria in enough quantities. Here we detail a method to measure mitochondrial respiratory chain activity and calcium transport in tobacco pollen tubes. ••
PubMed: 31406686
DOI: 10.1016/j.mex.2019.07.023 -
Journal of Applied Crystallography Apr 2020Digitonin has long been used as a mild detergent for extracting proteins from membranes for structure and function studies. As supplied commercially, digitonin is...
Digitonin has long been used as a mild detergent for extracting proteins from membranes for structure and function studies. As supplied commercially, digitonin is inhomogeneous and requires lengthy pre-treatment for reliable downstream use. Glyco-diosgenin (GDN) is a recently introduced synthetic surfactant with features that mimic digitonin. It is available in homogeneously pure form. GDN is proving to be a useful detergent, particularly in the area of single-particle cryo-electron microscopic studies of membrane integral proteins. With a view to using it as a detergent for crystallization trials by the or lipid cubic phase method, it was important to establish the carrying capacity of the cubic mesophase for GDN. This was quantified in the current study using small-angle X-ray scattering for mesophase identification and phase microstructure characterization as a function of temperature and GDN concentration. The data show that the lipid cubic phase formed by hydrated monoolein tolerates GDN to concentrations orders of magnitude in excess of those used for membrane protein studies. Thus, having GDN in a typical membrane protein preparation should not deter use of the method for crystallogenesis.
PubMed: 32280324
DOI: 10.1107/S1600576720002289 -
Chemico-biological Interactions Nov 2022Nuclear receptor pregnane X receptor (PXR) can induce significant liver enlargement through hepatocyte hypertrophy and proliferation. A previous report showed that...
Nuclear receptor pregnane X receptor (PXR) can induce significant liver enlargement through hepatocyte hypertrophy and proliferation. A previous report showed that during the process of PXR-induced liver enlargement, hepatocyte hypertrophy occurs around the central vein (CV) area while hepatocyte proliferation occurs around the portal vein (PV) area. However, the features of this spatial change remain unclear. Therefore, this study aims to explore the features of the spatial changes in hepatocytes in PXR-induced liver enlargement. PXR-induced spatial changes in hepatocyte hypertrophy and proliferation were confirmed in C57BL/6 mice. The liver was perfused with digitonin to destroy the hepatocytes around the CV or PV areas, and then the regional expression of proteins related to hepatocyte hypertrophy and proliferation was further measured. The results showed that the expression of PXR downstream proteins, such as cytochrome P450 (CYP) 3A11, CYP2B10, P-glycoprotein (P-gp) and organ anion transporting polypeptide 4 (OATP4) was upregulated around the CV area, while the expression of proliferation-related proteins such as cyclin B1 (CCNB1), cyclin D1 (CCND1) and serine/threonine NIMA-related kinase 2 (NEK2) was upregulated around the PV area. At the same time, the expression of cyclin-dependent kinase inhibitors such as retinoblastoma-like protein 2 (RBL2), cyclin-dependent kinase inhibitor 1B (CDKN1B) and CDKN1A was downregulated around the PV area. This study demonstrated that the spatial change in PXR-induced hepatocyte hypertrophy and proliferation is associated with the regional expression of PXR downstream targets and proliferation-related proteins and the regional distribution of triglycerides (TGs). These findings provide new insight into the understanding of PXR-induced hepatomegaly.
Topics: ATP Binding Cassette Transporter, Subfamily B; Animals; Anions; Cell Proliferation; Cyclin B1; Cyclin D1; Cyclin-Dependent Kinase Inhibitor p27; Cyclin-Dependent Kinases; Cytochrome P-450 CYP3A; Cytochrome P-450 Enzyme System; Digitonin; Hepatocytes; Hepatomegaly; Hypertrophy; Liver; Mice; Mice, Inbred C57BL; NIMA-Related Kinases; Pregnane X Receptor; Receptors, Cytoplasmic and Nuclear; Receptors, Steroid; Retinoblastoma-Like Protein p130; Serine; Threonine; Triglycerides
PubMed: 36030841
DOI: 10.1016/j.cbi.2022.110133 -
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 -
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 -
Veterinary Microbiology Apr 2024This study demonstrates for the first time that the matrix (M) protein of BEFV is a nuclear targeting protein that shuttles between the nucleus and the cytoplasm in a...
This study demonstrates for the first time that the matrix (M) protein of BEFV is a nuclear targeting protein that shuttles between the nucleus and the cytoplasm in a transcription-, carrier-, and energy-dependent manner. Experiments performed in both intact cells and digitonin-permeabilized cells revealed that M protein targets the nucleolus and requires carrier, cytosolic factors or energy input. By employing sequence and mutagenesis analyses, we have determined both nuclear localization signal (NLS) KKGKSK and nuclear export signal (NES) LIITSYL TI of M protein that are important for the nucleocytoplasmic shuttling of M protein. Furthermore, we found that both lamin A/C and chromosome maintenance region 1 (CRM-1) proteins could be coimmunoprecipitated and colocalized with the BEFV M protein. Knockdown of lamin A/C by shRNA and inhibition of CRM-1 by leptomycin B significantly reduced virus yield. Collectively, this study provides novel insights into nucleocytoplasmic shuttling of the BEFV M protein modulated by lamin A/C and CRM-1 and by a transcription- and carrier- and energy-dependent pathway.
Topics: Animals; Active Transport, Cell Nucleus; Cell Nucleus; Chromosomes; Cytoplasm; Lamin Type A; Nuclear Localization Signals; Ephemeral Fever Virus, Bovine; Viral Structural Proteins
PubMed: 38364467
DOI: 10.1016/j.vetmic.2024.110026 -
Synthetic Biology (Oxford, England) 2022Cell-free expression (CFE) systems are one of the main platforms for building synthetic cells. A major drawback is the orthogonality of cell-free systems across species....
Cell-free expression (CFE) systems are one of the main platforms for building synthetic cells. A major drawback is the orthogonality of cell-free systems across species. To generate a CFE system compatible with recently established minimal cell constructs, we attempted to optimize a bacterium-based CFE system using lysates of the genome-minimized cell JCVI-syn3A (Syn3A) and its close phylogenetic relative (Mcap). To produce mycoplasma-derived crude lysates, we systematically tested methods commonly used for bacteria, based on the S30 protocol of . Unexpectedly, after numerous attempts to optimize lysate production methods or composition of feeding buffer, none of the Mcap or Syn3A lysates supported cell-free gene expression. Only modest levels of transcription of RNA aptamers were observed. While our experimental systems were intended to perform transcription and translation, our assays focused on RNA. Further investigations identified persistently high ribonuclease (RNase) activity in all lysates, despite removal of recognizable nucleases from the respective genomes and attempts to inhibit nuclease activities in assorted CFE preparations. An alternative method using digitonin to permeabilize the mycoplasma cell membrane produced a lysate with diminished RNase activity yet still was unable to support cell-free gene expression. We found that intact mycoplasma cells poisoned cell-free extracts by degrading ribosomal RNAs, indicating that the mycoplasma cells, even the minimal cell, have a surface-associated RNase activity. However, it is not clear which gene encodes the RNase. This work summarizes attempts to produce mycoplasma-based CFE and serves as a cautionary tale for researchers entering this field. Graphical Abstract.
PubMed: 35774105
DOI: 10.1093/synbio/ysac008 -
Journal of Immunology (Baltimore, Md. :... May 2023Hepatic innate immune function plays an important role in the pathogenesis of many diseases. Importantly, a growing body of literature has firmly established the spatial...
Hepatic innate immune function plays an important role in the pathogenesis of many diseases. Importantly, a growing body of literature has firmly established the spatial heterogeneity of hepatocyte metabolic function; however, whether innate immune function is zonated remains unknown. To test this question, we exposed adult C57BL/6 mice to endotoxemia, and hepatic tissue was assessed for the acute phase response (APR). The zone-specific APR was evaluated in periportal and pericentral/centrilobular hepatocytes isolated using digitonin perfusion and on hepatic tissue using RNAscope and immunohistochemistry. Western blot, EMSA, chromatin immunoprecipitation, and immunohistochemistry were used to determine the role of the transcription factor NF-κB in mediating hepatic C-reactive protein (CRP) expression. Finally, the ability of mice lacking the NF-κB subunit p50 (p50-/-) to raise a hepatic APR was evaluated. We found that endotoxemia induces a hepatocyte transcriptional APR in both male and female mice, with Crp, Apcs, Fga, Hp, and Lbp expression being enriched in pericentral/centrilobular hepatocytes. Focusing our work on CRP expression, we determined that NF-κB transcription factor subunit p50 binds to consensus sequence elements present in the murine CRP promoter. Furthermore, pericentral/centrilobular hepatocyte p50 nuclear translocation is temporally associated with zone-specific APR during endotoxemia. Lastly, the APR and CRP expression is blunted in endotoxemic p50-/- mice. These results demonstrate that the murine hepatocyte innate immune response to endotoxemia includes zone-specific activation of transcription factors and target gene expression. These results support further study of zone-specific hepatocyte innate immunity and its role in the development of various disease states.
Topics: Male; Female; Animals; Mice; NF-kappa B; C-Reactive Protein; Endotoxemia; Mice, Inbred C57BL; Liver; NF-kappa B p50 Subunit; Immunity, Innate
PubMed: 36946778
DOI: 10.4049/jimmunol.2200900 -
Biochimica Et Biophysica Acta.... Apr 2023The FF-ATP synthase uses the energy stored in the electrochemical proton gradient to synthesize ATP. This complex is found in the inner mitochondrial membrane as a...
Deletion of the ATP20 gene in Ustilago maydis produces an unstable dimer of FF-ATP synthase associated with a decrease in mitochondrial ATP synthesis and a high HO production.
The FF-ATP synthase uses the energy stored in the electrochemical proton gradient to synthesize ATP. This complex is found in the inner mitochondrial membrane as a monomer and dimer. The dimer shows higher ATPase activity than the monomer and is essential for cristae folding. The monomer-monomer interface is constituted by subunits a, i/j, e, g, and k. The role of the subunit g in a strict respiratory organism is unknown. A gene knockout was generated in Ustilago maydis to study the role of subunit g on mitochondrial metabolism and cristae architecture. Deletion of the ATP20 gene, encoding the g subunit, did not affect cell growth or glucose consumption, but biomass production was lower in the mutant strain (gΔ strain). Ultrastructure observations showed that mitochondrial size and cristae shape were similar in wild-type and gΔ strains. The mitochondrial membrane potential in both strains had a similar magnitude, but oxygen consumption was higher in the WT strain. ATP synthesis was 20 % lower in the gΔ strain. Additionally, the mutant strain expressed the alternative oxidase in the early stages of growth (exponential phase), probably as a response to ROS stress. Dimer from mutant strain was unstable to digitonin solubilization, avoiding its isolation and kinetic characterization. The isolated monomeric state activated by n-dodecyl-β-D-maltopyranoside showed similar kinetic constants to the monomer from the WT strain. A decrease in mitochondrial ATP synthesis and the presence of the AOX during the exponential growth phase suggests that deletion of the g gene induces ROS stress.
Topics: Hydrogen Peroxide; Mitochondrial Proton-Translocating ATPases; Reactive Oxygen Species; Adenosine Triphosphate
PubMed: 36509127
DOI: 10.1016/j.bbabio.2022.148950 -
Biochimica Et Biophysica Acta.... Jul 2024Ginsenoside Rh2 (Rh2) is a ginseng saponin comprising a triterpene core and one unit of glucose and has attracted much attention due to its diverse biological...
Ginsenoside Rh2 (Rh2) is a ginseng saponin comprising a triterpene core and one unit of glucose and has attracted much attention due to its diverse biological activities. In the present study, we used small-angle X-ray diffraction, solid-state NMR, fluorescence microscopy, and MD simulations to investigate the molecular interaction of Rh2 with membrane lipids in the liquid-disordered (Ld) phase mainly composed of palmitoyloleoylphosphatidylcholine compared with those in liquid-ordered (Lo) phase mainly composed of sphingomyelin and cholesterol. The electron density profiles determined by X-ray diffraction patterns indicated that Rh2 tends to be present in the shallow interior of the bilayer in the Ld phase, while Rh2 accumulation was significantly smaller in the Lo phase. Order parameters at intermediate depths in the bilayer leaflet obtained from H NMR spectra and MD simulations indicated that Rh2 reduces the order of the acyl chains of lipids in the Ld phase. The dihydroxy group and glucose moiety at both ends of the hydrophobic triterpene core of Rh2 cause tilting of the molecular axis relative to the membrane normal, which may enhance membrane permeability by loosening the packing of lipid acyl chains. These features of Rh2 are distinct from steroidal saponins such as digitonin and dioscin, which exert strong membrane-disrupting activity.
PubMed: 38960300
DOI: 10.1016/j.bbamem.2024.184366