scholarly journals Retrograde Analysis of Calcium Signaling by CaMPARI2 Shows Cytosolic Calcium in Chondrocytes Is Unaffected by Parabolic Flights

Biomedicines ◽  
2022 ◽  
Vol 10 (1) ◽  
pp. 138
Author(s):  
Andreas Hammer ◽  
Geraldine Cerretti ◽  
Dario A. Ricciardi ◽  
David Schiffmann ◽  
Simon Maranda ◽  
...  
Keyword(s):  
Parabolic Flight ◽  
Microscopic Analysis ◽  
Cytosolic Calcium ◽  
Excellent Performance ◽  
Spatiotemporal Resolution ◽  
Cellular Processes ◽  
Secondary Messenger ◽  
Experiment Hardware ◽  
External Stimuli ◽  
Led Arrays

Calcium (Ca2+) elevation is an essential secondary messenger in many cellular processes, including disease progression and adaptation to external stimuli, e.g., gravitational load. Therefore, mapping and quantifying Ca2+ signaling with a high spatiotemporal resolution is a key challenge. However, particularly on microgravity platforms, experiment time is limited, allowing only a small number of replicates. Furthermore, experiment hardware is exposed to changes in gravity levels, causing experimental artifacts unless appropriately controlled. We introduce a new experimental setup based on the fluorescent Ca2+ reporter CaMPARI2, onboard LED arrays, and subsequent microscopic analysis on the ground. This setup allows for higher throughput and accuracy due to its retrograde nature. The excellent performance of CaMPARI2 was demonstrated with human chondrocytes during the 75th ESA parabolic flight campaign. CaMPARI2 revealed a strong Ca2+ response triggered by histamine but was not affected by the alternating gravitational load of a parabolic flight.

scholarly journals Advances and Perspectives in Chemical Imaging in Cellular Environments Using Electrochemical Methods

2018 ◽  
Author(s):  
Robert A. Lazenby ◽  
Ryan J. White
Keyword(s):  
Spatial Resolution ◽  
Chemical Imaging ◽  
Active Species ◽  
Electrochemical Methods ◽  
High Time Resolution ◽  
Spatiotemporal Resolution ◽  
Cellular Processes ◽  
Significant Research ◽  
Technological Developments ◽  
Multiple Frame

This review discusses a broad range of recent advances (2013-2017) of chemical imaging using electrochemical methods, with a particular focus on techniques that have been applied to study cellular processes, or techniques that show promise for use in this field in the future. Non-scanning techniques such as microelectrode arrays (MEAs) offer high time-resolution (< 10 ms) imaging, however at reduced spatial resolution. In contrast, scanning electrochemical probe microscopies (SEPMs) offer higher spatial resolution (as low as a few nm per pixel) imaging, with images collected typically over many minutes. Recent significant research efforts to improve the spatial resolution of SEPMs using nanoscale probes, and to improve the temporal resolution using fast scanning have resulted in movie (multiple frame) imaging with frame rates as low as a few seconds per image. Many SEPM techniques lack chemical specificity or have poor selectivity (defined by the choice of applied potential for redox-active species). This can be improved using multifunctional probes, ion-selective electrodes and tip-integrated biosensors, although additional effort may be required to preserve sensor performance after miniaturization of these probes. We discuss advances to the field of electrochemical imaging, and technological developments which are anticipated to extend the range of processes that can be studied. This includes imaging cellular processes with increased sensor selectivity and at much improved spatiotemporal resolution than has been previously customary.

scholarly journals Calcium ionophore, A23187, induces commitment to differentiation but inhibits the subsequent expression of erythroid genes in murine erythroleukemia cells

Blood ◽  
1991 ◽  
Vol 77 (6) ◽  
pp. 1362-1370 ◽  
Author(s):  
JO Hensold ◽  
G Dubyak ◽  
DE Housman
Keyword(s):  
Transcriptional Activation ◽  
Calcium Ionophore ◽  
Erythroid Differentiation ◽  
Cytosolic Calcium ◽  
Calcium Ionophore A23187 ◽  
Ionophore A23187 ◽  
Cellular Processes ◽  
Cellular Events ◽  
Erythroleukemia Cells ◽  
Murine Erythroleukemia

Abstract Murine erythroleukemia (MEL) cells are a useful model for studying the processes that regulate erythroid differentiation because exposure of these cells to a variety of chemical inducing agents results in expression of erythroid-specific genes and the resultant loss of cellular immortality. Previously it has been suggested that the calcium ionophore, A23187, has effects on the early cellular events that lead to the commitment of these cells to differentiation, but was not in itself sufficient to induce differentiation. We demonstrate here that A23187, as well as another calcium ionophore, ionomycin, are capable of inducing commitment to differentiation. Unlike other inducing agents, continual exposure to A23187 inhibits transcription of the erythroid- specific genes, beta-globin and Band 3. This effect is not attributable to an increase in cytosolic calcium concentration, because cells induced by ionomycin produce normal amounts of hemoglobin. These effects of A23187 on MEL cells confirm that commitment to differentiation is a distinct event from the subsequent transcriptional activation of erythroid genes. The ability of both ionophores to induce commitment to differentiation suggests that an increase in cytosolic calcium can trigger commitment to differentiation. These agents should prove useful in investigating the cellular processes that are responsible for commitment to differentiation.

scholarly journals Selective Target Inactivation Rather than Global Metabolic Dormancy Causes Antibiotic Tolerance in Uropathogens

2014 ◽  
Vol 58 (4) ◽  
pp. 2089-2097 ◽  
Author(s):  
Lee W. Goneau ◽  
Nigel S. Yeoh ◽  
Kyle W. MacDonald ◽  
Peter A. Cadieux ◽  
Jeremy P. Burton ◽  
...  
Keyword(s):  
Differential Susceptibility ◽  
Type I ◽  
Antibiotic Tolerance ◽  
Persister Cells ◽  
Content Type ◽  
E Coli ◽  
Cellular Processes ◽  
Multidrug Tolerance ◽  
External Stimuli ◽  
Susceptibility Patterns

ABSTRACTPersister cells represent a multidrug-tolerant (MDT), physiologically distinct subpopulation of bacteria. The ability of these organisms to survive lethal antibiotic doses raises concern over their potential role in chronic disease, such as recurrent urinary tract infection (RUTI). Persistence is believed to be conveyed through global metabolic dormancy, which yields organisms unresponsive to external stimuli. However, recent studies have contested this stance. Here, various antibiotics that target different cellular processes were used to dissect the activity of transcription, translation, and peptidoglycan turnover in persister cells. Differential susceptibility patterns were found in type I and type II persisters, and responses differed betweenStaphylococcus saprophyticusandEscherichia coliuropathogens. Further, SOS-deficient strains were sensitized to ciprofloxacin, suggesting DNA gyrase activity in persisters and indicating the importance of active DNA repair systems for ciprofloxacin tolerance. These results indicate that global dormancyper secannot sufficiently account for antibiotic tolerance. Rather, the activity of individual cellular processes dictates multidrug tolerance in an antibiotic-specific fashion. Furthermore, the susceptibility patterns of persisters depended on their mechanisms of onset, with subinhibitory antibiotic pretreatments selectively shutting down cognate targets and increasing the persister fraction against the same agent. Interestingly, antibiotics targeting transcription and translation enhanced persistence against multiple agents indirectly related to these processes. Conducting these assays with uropathogenicE. coliisolated from RUTI patients revealed an enriched persister fraction compared to organisms cleared with standard antibiotic therapy. This finding suggests that persister traits are either selected for during prolonged antibiotic treatment or initially contribute to therapy failure.

scholarly journals Calcium ionophore, A23187, induces commitment to differentiation but inhibits the subsequent expression of erythroid genes in murine erythroleukemia cells

Blood ◽  
1991 ◽  
Vol 77 (6) ◽  
pp. 1362-1370 ◽  
Author(s):  
JO Hensold ◽  
G Dubyak ◽  
DE Housman
Keyword(s):  
Transcriptional Activation ◽  
Calcium Ionophore ◽  
Erythroid Differentiation ◽  
Cytosolic Calcium ◽  
Calcium Ionophore A23187 ◽  
Ionophore A23187 ◽  
Cellular Processes ◽  
Cellular Events ◽  
Erythroleukemia Cells ◽  
Murine Erythroleukemia

Murine erythroleukemia (MEL) cells are a useful model for studying the processes that regulate erythroid differentiation because exposure of these cells to a variety of chemical inducing agents results in expression of erythroid-specific genes and the resultant loss of cellular immortality. Previously it has been suggested that the calcium ionophore, A23187, has effects on the early cellular events that lead to the commitment of these cells to differentiation, but was not in itself sufficient to induce differentiation. We demonstrate here that A23187, as well as another calcium ionophore, ionomycin, are capable of inducing commitment to differentiation. Unlike other inducing agents, continual exposure to A23187 inhibits transcription of the erythroid- specific genes, beta-globin and Band 3. This effect is not attributable to an increase in cytosolic calcium concentration, because cells induced by ionomycin produce normal amounts of hemoglobin. These effects of A23187 on MEL cells confirm that commitment to differentiation is a distinct event from the subsequent transcriptional activation of erythroid genes. The ability of both ionophores to induce commitment to differentiation suggests that an increase in cytosolic calcium can trigger commitment to differentiation. These agents should prove useful in investigating the cellular processes that are responsible for commitment to differentiation.

Ryanodine receptors mediate high intracellular Ca2+ and some myocyte damage following eccentric contractions in rat fast twitch skeletal muscle

2021 ◽  
Author(s):  
Ayaka Tabuchi ◽  
Yoshinori Tanaka ◽  
Ryo Takagi ◽  
Hideki Shirakawa ◽  
Tsubasa Shibaguchi ◽  
...  
Keyword(s):  
Muscle Damage ◽  
Ryanodine Receptors ◽  
Cytosolic Calcium ◽  
Specific Pattern ◽  
Eccentric Contractions ◽  
Cellular Processes ◽  
Area 5 ◽  
Myocyte Damage ◽  
Fast Twitch

Eccentric contractions (ECC) facilitate cytosolic calcium ion (Ca2+) release from the sarcoplasmic reticulum (SR) and Ca2+ influx from extracellular space. Ca2+ is a vital signaling messenger that regulates multiple cellular processes via its spatial and temporal concentration ([Ca2+]i) dynamics. We hypothesized that: 1) a specific pattern of spatial/temporal intramyocyte Ca2+ dynamics portends muscle damage following ECC, and 2) these dynamics would be regulated by the ryanodine receptor (RyR). [Ca2+]i in the tibialis anterior muscles of anesthetized adult Wistar rats was measured by ratiometric (i.e. ratio, R, 340/380 nm excitation) in vivo bioimaging with Fura-2 pre-ECC and at 5 and 24 hours post-ECC (5 x 40 contractions). Rats received RyR inhibitor dantrolene (DAN; 10 mg/kg i.p.) immediately post-ECC (+DAN). Muscle damage was evaluated by histological analysis on hematoxylin-eosin stained muscle sections. Compared to control (CONT, no ECC), [Ca2+]i distribution was heterogeneous with increased % total area of high [Ca2+]i sites (operationally defined as R > 1.39 i.e., > 1 SD of mean control) 5 hours post-ECC (CONT, 14.0 ± 8.0; ECC5h: 52.0 ± 7.4%, p < 0.01). DAN substantially reduced the high [Ca2+]i area 5 hours post-ECC (ECC5h+DAN: 6.4 ± 3.1%, p < 0.01) and myocyte damage (ECC24h, 63.2 ± 1.0%; ECC24h+DAN, 29.1 ± 2.2%, p < 0.01). Temporal and spatially-amplified [Ca2+]i fluctuations occurred regardless of DAN (ECC vs ECC+DAN, p > 0.05). These results suggest that the RyR-mediated local high [Ca2+]i itself is related to the magnitude of muscle damage while the [Ca2+]i fluctuation is an RyR-independent phenomenon.

Validamycin A enhances the interaction between neutral trehalase and 14‑3‑3 protein Bmh1 in Fusarium graminearum

2021 ◽  
Author(s):  
Li Ren ◽  
Yi-Ping Hou ◽  
Yuanye Zhu ◽  
Fei-Fei Zhao ◽  
Yabing Duan ◽  
...  
Keyword(s):  
High Temperature ◽  
Fusarium Graminearum ◽  
Protein Localization ◽  
Enzyme Activation ◽  
Hog Pathway ◽  
Temperature Stresses ◽  
Validamycin A ◽  
Cellular Processes ◽  
External Stresses ◽  
External Stimuli

Trehalase is considered the main target of the biological fungicide validamycin A, and toxicology mechanism of validamycin A is unknown. 14-3-3 proteins, highly conserved proteins, participate in diverse cellular processes, including enzyme activation, protein localization and molecular chaperone. In Saccharomyces cerevisiae, the 14-3-3 protein Bmh1could interact with Nth1 to respond specific external stimuli. Here, we characterized FgNth, FgBmh1, and FgBmh2 in Fusarium graminearum. ΔFgNth, ΔFgBmh1, and ΔFgBmh2 displayed great growth defects when compared to wild-type PH-1. When exposed to validamycin A, high osmotic and high temperature stresses, ΔFgNth, ΔFgBmh1, and ΔFgBmh2 showed more tolerance than WT. Both ΔFgNth and ΔFgBmh1 displayed reduced deoxynivalenol (DON) production but opposite for ΔFgBmh2, and all three deletion mutants showed reduced virulence on wheat coleoptiles. In addition, Co-immunoprecipitation (Co-IP) experiments suggested that FgBmh1 and FgBmh2 both interact with FgNth, but no interaction was detected between FgBmh1 and FgBmh2 in our experiments. Further, validamycin A enhances the interaction between FgBmh1 and FgNth in a positive correlation under concentrations of 1-100μg/mL. Besides, both high osmotic and high temperature stresses promote the interaction between FgBmh1 and FgNth. Co-IP assay also showed that neither FgBmh1 nor FgBmh2 could interact with FgPbs2, a MAPKK kinase in the high-osmolarity glycerol (HOG) pathway. However, FgBmh2 but not FgBmh1 binds to the heat shock protein FgHsp70 in F. graminearum. Taken together, our results demonstrate that FgNth and FgBmhs are involved in growth, responces to external stresses and virulence, and validamycin A enhanced the interaction between FgNth and FgBmh1in F. graminearum.

scholarly journals Dietary, Metabolic, and Potentially Environmental Modulation of the Lysine Acetylation Machinery

2010 ◽  
Vol 2010 ◽  
pp. 1-14 ◽  
Author(s):  
Go-Woon Kim ◽  
Goran Gocevski ◽  
Chao-Jung Wu ◽  
Xiang-Jiao Yang
Keyword(s):  
Expression Patterns ◽  
Chromatin Dynamics ◽  
Cellular Processes ◽  
The Past ◽  
Pathological Conditions ◽  
Good State ◽  
Therapeutic Development ◽  
Dietary Components ◽  
External Stimuli

Healthy lifestyles and environment produce a good state of health. A number of scientific studies support the notion that external stimuli regulate an individual's epigenomic profile. Epigenetic changes play a key role in defining gene expression patterns under both normal and pathological conditions. As a major posttranslational modification, lysine (K) acetylation has received much attention, owing largely to its significant effects on chromatin dynamics and other cellular processes across species. Lysine acetyltransferases and deacetylases, two opposing families of enzymes governing K-acetylation, have been intimately linked to cancer and other diseases. These enzymes have been pursued by vigorous efforts for therapeutic development in the past 15 years or so. Interestingly, certain dietary components have been found to modulate acetylation levelsin vivo. Here we review dietary, metabolic, and environmental modulators of the K-acetylation machinery and discuss how they may be of potential value in the context of disease prevention.

scholarly journals Dynamic mRNP Remodeling in Response to Internal and External Stimuli

Biomolecules ◽  
2020 ◽  
Vol 10 (9) ◽  
pp. 1310 ◽  
Author(s):  
Kathi Zarnack ◽  
Sureshkumar Balasubramanian ◽  
Michael P. Gantier ◽  
Vladislav Kunetsky ◽  
Michael Kracht ◽  
...  
Keyword(s):  
Gene Expression ◽  
Signal Transduction ◽  
Rna Binding ◽  
Environmental Changes ◽  
Rna Binding Proteins ◽  
Regulation Of Gene Expression ◽  
Cellular Processes ◽  
Messenger Ribonucleoprotein ◽  
Transcriptional Modulation ◽  
External Stimuli

Signal transduction and the regulation of gene expression are fundamental processes in every cell. RNA-binding proteins (RBPs) play a key role in the post-transcriptional modulation of gene expression in response to both internal and external stimuli. However, how signaling pathways regulate the assembly of RBPs with mRNAs remains largely unknown. Here, we summarize observations showing that the formation and composition of messenger ribonucleoprotein particles (mRNPs) is dynamically remodeled in space and time by specific signaling cascades and the resulting post-translational modifications. The integration of signaling events with gene expression is key to the rapid adaptation of cells to environmental changes and stress. Only a combined approach analyzing the signal transduction pathways and the changes in post-transcriptional gene expression they cause will unravel the mechanisms coordinating these important cellular processes.

scholarly journals A pharmacological cocktail for arresting actin dynamics in living cells

2011 ◽  
Vol 22 (21) ◽  
pp. 3986-3994 ◽  
Author(s):  
Grace E. Peng ◽  
Sarah R. Wilson ◽  
Orion D. Weiner
Keyword(s):  
Leading Edge ◽  
Actin Dynamics ◽  
Actin Assembly ◽  
Actin Reorganization ◽  
Cellular Processes ◽  
Wide Range ◽  
Morphological Polarity ◽  
Nih 3T3 ◽  
External Stimuli

The actin cytoskeleton is regulated by factors that influence polymer assembly, disassembly, and network rearrangement. Drugs that inhibit these events have been used to test the role of actin dynamics in a wide range of cellular processes. Previous methods of arresting actin rearrangements take minutes to act and work well in some contexts, but can lead to significant actin reorganization in cells with rapid actin dynamics, such as neutrophils. In this paper, we report a pharmacological cocktail that not only arrests actin dynamics but also preserves the structure of the existing actin network in neutrophil-like HL-60 cells, human fibrosarcoma HT1080 cells, and mouse NIH 3T3 fibroblast cells. Our cocktail induces an arrest of actin dynamics that initiates within seconds and persists for longer than 10 min, during which time cells maintain their responsivity to external stimuli. With this cocktail, we demonstrate that actin dynamics, and not simply morphological polarity or actin accumulation at the leading edge, are required for the spatial persistence of Rac activation in HL-60 cells. Our drug combination preserves the structure of the existing cytoskeleton while blocking actin assembly, disassembly, and rearrangement, and should prove useful for investigating the role of actin dynamics in a wide range of cellular signaling contexts.

scholarly journals A Complete and Low-Cost Cardiac Optical Mapping System in Translational Animal Models

2021 ◽  
Vol 12 ◽  
Author(s):  
Manuel Marina-Breysse ◽  
Alba García-Escolano ◽  
Joaquín Vila-García ◽  
Gabriel Reale-Nosei ◽  
José M. Alfonso-Almazán ◽  
...  
Keyword(s):  
Action Potentials ◽  
Cardiac Electrophysiology ◽  
High Speed ◽  
Optical Mapping ◽  
Low Cost ◽  
Cytosolic Calcium ◽  
Calcium Transients ◽  
Spatiotemporal Resolution ◽  
Mapping System ◽  
Computer Scientists

Clinicians, biologists, physicists, engineers, and computer scientists are coming together to better understand heart disease, which is currently the leading cause of death globally. Optical mapping, a high-speed fluorescence imaging technique that visualizes and measures key cardiac parameters such as action potentials, cytosolic calcium transients, and fibrillation dynamics, is a core research tool that has arisen from such interdisciplinary collaborations. In an effort to broaden its use, especially among clinical scientists and students, we developed a complete and low-cost optical mapping system, including a constant-flow Langendorff perfusion system, which minimizes the economic threshold to widespread use of this powerful tool in cardiac electrophysiology research. The system described here provides high spatiotemporal resolution data about action potentials, intracellular calcium transients and fibrillation wave dynamics in isolated Langendorff-perfused hearts (pigs and rabbits), relevant for translational research. All system components and software elements are fully disclosed with the aim of increasing the use of this affordable and highly versatile tool among clinicians, basic scientists and students wishing to tackle their own research questions with their own customizable systems.

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