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Proceedings of the National Academy of... Jan 1997According to the free radical theory of aging, reactive oxygen species cause oxidative damage, proposed to be an underlying factor of the aging process. In the current...
According to the free radical theory of aging, reactive oxygen species cause oxidative damage, proposed to be an underlying factor of the aging process. In the current study, we have used electron paramagnetic resonance spin labeling, measurements of protein carbonyl content, an index of protein oxidation, and determination of the activity of glutamine synthetase (an oxidatively sensitive enzyme) to report that cortical synaptosomal membranes from the senescence accelerated-prone (SAMP8) mouse showed structural characteristics of free radical oxidative stress relative to the senescence accelerated-resistant (SAMR1) mouse. The SAMP8 mouse exhibited a decrease in the relevant EPR parameter consistent with oxidative stress (P < 0.002), a decreased glutamine synthetase activity (P < 0.05), and an increased protein carbonyl content (P < 0.01) compared with these parameters in the SAMR1 mouse. Further, because free radical trapping compounds have been demonstrated to extend maximum life span and improve cognition in SAMP8 mice, we investigated the protective nature of the known free radical scavenger, N-tert-butyl-alpha-phenylnitrone (PBN), on the physical state of cortical synaptosomal membrane proteins. For 14 days, SAMR1 and SAMP8 mice were injected with 30 mg/kg PBN while the controls were injected with the corresponding volume of saline. Characteristic of less oxidized systems, cortical synaptosomal membranes from the PBN-injected SAMP8 mouse exhibited a return toward normal values of the relevant EPR parameter [the M1 = +1 low-field weakly immobilized line/M1 = +1 low-field strongly immobilized line (W/S) ratio of a protein-specific spin label] (P < 0.001) compared with that from saline-injected SAMP8 mice. In SAMR1 mice, in contrast to SAMP8, there was no significant change in the conformation of membrane proteins or protein carbonyl content of cortical synaptosomal membranes from the PBN-injected and saline-injected SAMR1 mice, showing that PBN itself did not induce conformational changes in cortical synaptosomal membrane proteins. The results are discussed with reference to the use of free radical scavengers as potential anti-aging agents.
Topics: Aging; Animals; Brain; Cytosol; Free Radicals; Membrane Proteins; Mice; Mice, Mutant Strains; Nerve Tissue Proteins; Oxidation-Reduction; Synaptosomes
PubMed: 9012843
DOI: 10.1073/pnas.94.2.674 -
Developmental Biology Apr 2008Fertilization activates development by stimulating a plethora of ATP consuming processes that must be provided for by an up-regulation of energy production in the...
Fertilization activates development by stimulating a plethora of ATP consuming processes that must be provided for by an up-regulation of energy production in the zygote. Sperm-triggered Ca(2+) oscillations are known to be responsible for the stimulation of both ATP consumption and ATP supply but the mechanism of up regulation of energy production at fertilization is still unclear. By measuring [Ca(2+)] and [ATP] in the mitochondria of fertilized mouse eggs we demonstrate that sperm entry triggers Ca(2+) oscillations in the cytosol that are transduced into mitochondrial Ca(2+) oscillations pacing mitochondrial ATP production. This results, during fertilization, in an increase in both [ATP](mito) and [ATP](cyto). We also observe the stimulation of ATP consumption accompanying fertilization by monitoring [Ca(2+)](cyto) and [ATP](cyto) during fertilization of starved eggs. Our observations reveal that lactate, in contrast to pyruvate, does not fuel mitochondrial ATP production in the zygote. Therefore lactate-derived pyruvate is somehow diverted from mitochondrial oxidation and may be channeled to other metabolic routes. Together with our earlier findings, this study confirms the essential role for exogenous pyruvate in the up-regulation of ATP production at the onset of development, and suggests that lactate, which does not fuel energetic metabolism may instead regulate the intracellular redox potential.
Topics: Adenosine Triphosphate; Animals; Cytosol; Female; Fluorescence; Homeostasis; Luminescence; Membrane Potentials; Mice; Mitochondria; Ovum; Zygote
PubMed: 18342302
DOI: 10.1016/j.ydbio.2008.02.004 -
Journal of Applied Physiology... Sep 2022
Topics: Calcium; Calcium Signaling; Cytosol; Heart
PubMed: 35981731
DOI: 10.1152/japplphysiol.00453.2022 -
Journal of the American Chemical Society Aug 2017Quantum dots (QDs) are extremely bright, photostable, nanometer particles broadly used to investigate single molecule dynamics in vitro. However, the use of QDs in vivo...
Quantum dots (QDs) are extremely bright, photostable, nanometer particles broadly used to investigate single molecule dynamics in vitro. However, the use of QDs in vivo to investigate single molecule dynamics is impaired by the absence of an efficient way to chemically deliver them into the cytosol of cells. Indeed, current methods (using cell-penetrating peptides for instance) provide very low yields: QDs stay at the plasma membrane or are trapped in endosomes. Here, we introduce a technology based on cell-penetrating poly(disulfide)s that solves this problem: we deliver about 70 QDs per cell, and 90% appear to freely diffuse in the cytosol. Furthermore, these QDs can be functionalized, carrying GFP or anti-GFP nanobodies for instance. Our technology thus paves the way toward single molecule imaging in cells and living animals, allowing to probe biophysical properties of the cytosol.
Topics: Animals; Cell Line; Cell-Penetrating Peptides; Cytosol; Disulfides; Drosophila; Drug Delivery Systems; Humans; Molecular Structure; Quantum Dots
PubMed: 28741941
DOI: 10.1021/jacs.7b02952 -
Free Radical Biology & Medicine Jan 2023Cytosolic and organelle redox are highly interrelated, and their alterations play critical roles in both physiological and pathological cell states. This highly...
Cytosolic and organelle redox are highly interrelated, and their alterations play critical roles in both physiological and pathological cell states. This highly regulated process is crucial in life-death decisions of cells. Among organelles, the mitochondrion is the major source of intracellular-ROS and contributes to oxidation damage-induced cell death. Increase in cytosolic-redox and mitochondrial-redox is evident in cells undergoing diverse forms of cell death, such as apoptosis, necrosis, and necroptosis. The hierarchical profiling of redox signaling at the cytosol and mitochondria in a single cell is important to understand the relative contribution of each species in the initiation and shaping of cell death. Here, we demonstrate the potential application of ratiometric redox GFP (roGFP) and intensity-based redox-sensitive RFP (rxRFP) targeted to mitochondria in revealing both rapid and slow progressing changes in redox during cell division and in cells undergoing multiple modes of cell death. To generate imaging quality signal, single-cell clones stably expressing both roGFP at the cytosol and rxRFP probes targeted to mitochondria were generated. The cells provided sufficient temporal resolution with imaging-ready signal for the real-time visualization of rapidly progressing redox alterations at the cytosol and mitochondria. The long-time imaging of the cells revealed that a moderate increase in cytosolic ROS marks the division stage. Similarly, distinct forms of cell death trigger a unique and temporally regulated redox change at the cytosol and mitochondria, suggesting the potential utility of the sensor cells to dissect the nature of cell death pathways induced by specific forms of stress.
Topics: Cytosol; Reactive Oxygen Species; Mitochondria; Oxidation-Reduction; Cell Death; Cell Division
PubMed: 36427748
DOI: 10.1016/j.freeradbiomed.2022.11.031 -
Biophysical Journal Jun 2007It is currently assumed that two or more pools of the same metabolite can coexist in the cytosolic compartment of mammalian cells. These pools are thought to be... (Comparative Study)
Comparative Study
It is currently assumed that two or more pools of the same metabolite can coexist in the cytosolic compartment of mammalian cells. These pools are thought to be generated by the differential subcellular location of enzymes and transporters, much in the way calcium microdomains arise by the combined workings of channels, buffers, and pumps. With the aim of estimating the amplitude and spatial dimensions of these metabolite pools, we developed an analytical tool based on Brownian diffusion and the turnover numbers of the proteins involved. The outcome of the analysis is that ATP, glucose, pyruvate, lactate, and glutamate cannot be concentrated at their sources to an extent that would affect their downstream targets. For these metabolites, and others produced by slow enzymes or transporters and present at micromolar concentrations or higher, the cytosol behaves as a well-mixed, homogenous compartment. In contrast, the analysis showed microdomains known to be generated by calcium channels and revealed that calcium and pH nanodomains are to be found in the vicinity of slow enzymes and transporters in the steady state. The analysis can be readily applied to any other molecule, provided knowledge is available about rate of production, average concentration, and diffusion coefficient. Our main conclusion is that the notion of cytosolic compartmentation of metabolites needs reevaluation, as it seems to be in conflict with the underlying physical chemistry.
Topics: Adenosine Triphosphate; Cytosol; Models, Biological
PubMed: 17369414
DOI: 10.1529/biophysj.106.100925 -
Environmental Health Perspectives Mar 1990Use of clonal strains of prolactin (PRL)- and growth hormone-producing rat pituitary cells has proven informative in elucidating a number of the early biochemical,... (Review)
Review
Use of clonal strains of prolactin (PRL)- and growth hormone-producing rat pituitary cells has proven informative in elucidating a number of the early biochemical, ionic, and secretory events regulated by the hypothalamic tripeptide, thyrotropin-releasing hormone (TRH). TRH causes biphasic changes in the concentration of cytosolic free calcium [( Ca2+]i) in GH4C1 cells and biphasic changes in hormone secretion. Early changes occur on a msecond to second scale and late changes, on a time scale of minutes. Although increases in [Ca2+]i are essential for enhanced secretion, at least in the case of the rapid initial phase, the TRH-induced increase in [Ca2+]i is necessary, but not sufficient to enhance secretion. A co-mediator with calcium appears to be diacylglycerol. The majority of the calcium involved in the early phase of rise in [Ca2+]i induced by TRH is derived from intracellular sources, while essentially all of the calcium rise observed in the late phase is derived from extracellular calcium entering the cell through both voltage-dependent and voltage-independent conductances. Because TRH causes an elevation of inositol(1,4,5) trisphosphate [Ins(1,4,5)P3] within seconds, but not mseconds, further studies are required before it can be concluded unequivocally that Ins(1,4,5)P3 is the sole mediator of the rapid phase of rise in [Ca2+]i induced by TRH in GH-cells.(ABSTRACT TRUNCATED AT 250 WORDS)
Topics: Animals; Calcium; Cells, Cultured; Cytosol; Hormones; Humans; Models, Biological
PubMed: 2190815
DOI: 10.1289/ehp.908427 -
Current Biology : CB Apr 2010Viruses are detected by the innate immune system, leading to the initiation of the anti-viral immune response via the production of type I interferons and inflammatory... (Review)
Review
Viruses are detected by the innate immune system, leading to the initiation of the anti-viral immune response via the production of type I interferons and inflammatory cytokines such as interleukin-1. Remarkable progress has been made over the past few years towards understanding the contribution of Toll-like receptors, RIG-I like receptors, NOD-like receptors and HIN-200 family members to viral detection. Furthermore, new complexities in the signaling pathways activated by these receptors continue to be revealed. Together, these new insights are leading to therapeutically useful information in the fight against viruses.
Topics: Animals; Cytosol; DNA, Viral; Humans; Immunity, Innate; Models, Immunological; Nod Signaling Adaptor Proteins; Receptors, Immunologic; Signal Transduction; Toll-Like Receptors; Viruses
PubMed: 20392426
DOI: 10.1016/j.cub.2010.01.044 -
BMC Biology Jan 2018Phthiocerol dimycocerosates (PDIM), glycolipids found on the outer surface of virulent members of the Mycobacterium tuberculosis (Mtb) complex, are a major contributing...
BACKGROUND
Phthiocerol dimycocerosates (PDIM), glycolipids found on the outer surface of virulent members of the Mycobacterium tuberculosis (Mtb) complex, are a major contributing factor to the pathogenesis of Mtb. Myelocytic cells, such as macrophages and dendritic cells, are the primary hosts for Mtb after infection and previous studies have shown multiple roles for PDIM in supporting Mtb in these cells. However, Mtb can infect other cell types. We previously showed that Mtb efficiently replicates in human lymphatic endothelial cells (hLECs) and that the hLEC cytosol acts as a reservoir for Mtb in humans. Here, we examined the role of PDIM in Mtb translocation to the cytosol in hLECs.
RESULTS
Analysis of a Mtb mutant unable to produce PDIM showed less co-localisation of bacteria with the membrane damage marker Galectin-8 (Gal8), indicating that PDIM strongly contribute to phagosomal membrane damage. Lack of this Mtb lipid also leads to a reduction in the proportion of Mtb co-localising with markers of macroautophagic removal of intracellular bacteria (xenophagy) such as ubiquitin, p62 and NDP52. hLEC imaging with transmission electron microscopy shows that Mtb mutants lacking PDIM are much less frequently localised in the cytosol, leading to a lower intracellular burden.
CONCLUSIONS
PDIM is needed for the disruption of the phagosome membrane in hLEC, helping Mtb avoid the hydrolytic phagolysosomal milieu. It facilitates the translocation of Mtb into the cytosol, and the decreased intracellular burden of Mtb lacking PDIM indicates that the cytosol is the preferred replicative niche for Mtb in these cells. We hypothesise that pharmacological targeting of PDIM synthesis in Mtb would reduce the formation of a lymphatic reservoir of Mtb in humans.
Topics: Cytosol; Endothelial Cells; Humans; Intracellular Fluid; Lipids; Mycobacterium tuberculosis; Phagocytosis
PubMed: 29325545
DOI: 10.1186/s12915-017-0471-6 -
Proceedings of the National Academy of... Oct 2017The ciliary localization of odorant receptors (ORs) is evolutionary conserved and essential for olfactory transduction. However, how the transport of ORs is regulated in...
The ciliary localization of odorant receptors (ORs) is evolutionary conserved and essential for olfactory transduction. However, how the transport of ORs is regulated in mammalian olfactory sensory neurons is poorly understood. Here we demonstrate that odorant responsiveness and OR transport is regulated by the Hedgehog pathway. OR transport is inhibited by conditional gene inactivation of the Hedgehog signal mediator Smoothened (Smo) as well as by systemic administration of the Smo inhibitor vismodegib, a clinically used anticancer drug reported to distort smell perception in patients. The ciliary phenotype of Smo inhibition is haploinsufficient, cell autonomous, and correlates with the accumulation of OR-containing putative transport vesicles in the cytosol. The Smo-dependent OR transport route works in parallel with a low basal transport of vesicle containing both ORs and other olfactory transduction components. These findings both define a physiological function of Hedgehog signaling in olfaction and provide an important evolutionary link between olfaction and the requirement of a ciliary compartment for Hedgehog signaling.
Topics: Anilides; Animals; Cilia; Cytosol; Hedgehog Proteins; Mice; Mice, Inbred C57BL; Protein Transport; Pyridines; Receptors, Odorant; Signal Transduction; Smoothened Receptor
PubMed: 29078327
DOI: 10.1073/pnas.1708321114