scholarly journals Biological cell-based screening for scientific membranal and cytoplasmatic markers using dielectric spectroscopy

2008 ◽  
Vol 2 (2) ◽  
pp. 111-121
Author(s):  
Ragini Raj Singh ◽  
◽  
Amit Ron ◽  
Nick Fishelson ◽  
Irena Shur ◽  
...  
Keyword(s):  
Cell Lines ◽  
Dielectric Spectroscopy ◽  
Cell Biology ◽  
Screening Tool ◽  
Excitation Frequency ◽  
Cell Physiology ◽  
Biological Cell ◽  
Non Invasive ◽  
Native Cell ◽  
Biology Research

Dielectric spectroscopy (DS) of living biological cells is based on the analysis of cells suspended in a physiological medium. It provides knowledge of the polarization-relaxation response of the cells to external electric field as function of the excitation frequency. This response is strongly affected by both structural and molecular properties of the cells and, therefore, can reveal rare insights into cell physiology and behaviour. This study demonstrates the mapping potential of DS after cytoplasmic and membranal markers for cell-based screening analysis. The effect of membrane permittivity and cytoplasm conductivity was examined using tagged MBA and MDCK cell lines respectively. The comparison of the dielectric spectra of tagged and native cell lines reveals clear differences between the cells. In addition, the differences in the matching dielectric properties of the cells were discovered. Those findings support the high distinction resolution and sensitivity of DS after fine molecular and cellular changes, and hence, highlight the high potential of DS as non invasive screening tool in cell biology research.

scholarly journals Do you know the sex of your cells?

2014 ◽  
Vol 306 (1) ◽  
pp. C3-C18 ◽  
Author(s):  
Kalpit Shah ◽  
Charles E. McCormack ◽  
Neil A. Bradbury
Keyword(s):  
Cell Lines ◽  
Sex Chromosomes ◽  
Cell Biology ◽  
Male Subject ◽  
Cell Physiology ◽  
Reproductive Tissue ◽  
Renal Cells ◽  
Pancreatic Acini ◽  
The Past ◽  
Prostate Cells

Do you know the sex of your cells? Not a question that is frequently heard around the lab bench, yet thanks to recent research is probably one that should be asked. It is self-evident that cervical epithelial cells would be derived from female tissue and prostate cells from a male subject (exemplified by HeLa and LnCaP, respectively), yet beyond these obvious examples, it would be true to say that the sex of cell lines derived from non-reproductive tissue, such as lung, intestine, kidney, for example, is given minimal if any thought. After all, what possible impact could the presence of a Y chromosome have on the biochemistry and cell biology of tissues such as the exocrine pancreatic acini? Intriguingly, recent evidence has suggested that far from being irrelevant, genes expressed on the sex chromosomes can have a marked impact on the biology of such diverse tissues as neurons and renal cells. It is also policy of AJP-Cell Physiology that the source of all cells utilized (species, sex, etc.) should be clearly indicated when submitting an article for publication, an instruction that is rarely followed ( http://www.the-aps.org/mm/Publications/Info-For-Authors/Composition ). In this review we discuss recent data arguing that the sex of cells being used in experiments can impact the cell's biology, and we provide a table outlining the sex of cell lines that have appeared in AJP-Cell Physiology over the past decade.

Expression of Urokinase and Its Receptor in Invasive and Non-Invasive Prostate Cancer Cell Lines

1992 ◽  
Vol 68 (06) ◽  
pp. 662-666 ◽  
Author(s):  
W Hollas ◽  
N Hoosein ◽  
L W K Chung ◽  
A Mazar ◽  
J Henkin ◽  
...  
Keyword(s):  
Prostate Cancer ◽  
Steady State ◽  
Cell Surface ◽  
Cell Lines ◽  
Cancer Cell ◽  
Prostate Cancer Cell ◽  
Cancer Cell Lines ◽  
Lncap Cells ◽  
Prostate Cancer Cell Lines ◽  
Non Invasive

SummaryWe previously reported that extracellular matrix invasion by the prostate cancer cell lines, PC-3 and DU-145 was contingent on endogenous urokinase being bound to a specific cell surface receptor. The present study was undertaken to characterize the expression of both urokinase and its receptor in the non-invasive LNCaP and the invasive PC-3 and DU-145 prostate cells. Northern blotting indicated that the invasive PC-3 cells, which secreted 10 times more urokinase (680 ng/ml per 106 cells per 48 h) than DU-145 cells (63 ng/ml per 106 cells per 48 h), had the most abundant transcript for the plasminogen activator. This, at least, partly reflected a 3 fold amplification of the urokinase gene in the PC-3 cells. In contrast, urokinase-specific transcript could not be detected in the non-invasive LNCaP cells previously characterized as being negative for urokinase protein. Southern blotting indicated that this was not a consequence of deletion of the urokinase gene. Crosslinking of radiolabelled aminoterminal fragment of urokinase to the cell surface indicated the presence of a 51 kDa receptor in extracts of the invasive PC-3 and DU-145 cells but not in extracts of the non-invasive LNCaP cells. The amount of binding protein correlated well with binding capacities calculated by Scatchard analysis. In contrast, the steady state level of urokinase receptor transcript was a poor predictor of receptor display. PC-3 cells, which were equipped with 25,000 receptors per cell had 2.5 fold more steady state transcript than DU-145 cells which displayed 93,000 binding sites per cell.

scholarly journals Conditionally immortalised leukaemia initiating cells co-expressing Hoxa9/Meis1 demonstrate microenvironmental adaptation properties ex vivo while maintaining myelomonocytic memory

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Maike Stahlhut ◽  
Teng Cheong Ha ◽  
Ekaterina Takmakova ◽  
Michael A. Morgan ◽  
Adrian Schwarzer ◽  
...  
Keyword(s):  
Stem Cell ◽  
Cell Fate ◽  
Cell Lines ◽  
Cell Biology ◽  
Ex Vivo ◽  
Haematopoietic Stem Cell ◽  
Transcriptional Dysregulation ◽  
Conditional Gene Expression ◽  
Exponential Expansion ◽  
Drug Dependent

AbstractRegulation of haematopoietic stem cell fate through conditional gene expression could improve understanding of healthy haematopoietic and leukaemia initiating cell (LIC) biology. We established conditionally immortalised myeloid progenitor cell lines co-expressing constitutive Hoxa9.EGFP and inducible Meis1.dTomato (H9M-ciMP) to study growth behaviour, immunophenotype and morphology under different cytokine/microenvironmental conditions ex vivo upon doxycycline (DOX) induction or removal. The vector design and drug-dependent selection approach identified new retroviral insertion (RVI) sites that potentially collaborate with Meis1/Hoxa9 and define H9M-ciMP fate. For most cell lines, myelomonocytic conditions supported reversible H9M-ciMP differentiation into neutrophils and macrophages with DOX-dependent modulation of Hoxa9/Meis1 and CD11b/Gr-1 expression. Here, up-regulation of Meis1/Hoxa9 promoted reconstitution of exponential expansion of immature H9M-ciMPs after DOX reapplication. Stem cell maintaining conditions supported selective H9M-ciMP exponential growth. H9M-ciMPs that had Ninj2 RVI and were cultured under myelomonocytic or stem cell maintaining conditions revealed the development of DOX-dependent acute myeloid leukaemia in a murine transplantation model. Transcriptional dysregulation of Ninj2 and distal genes surrounding RVI (Rad52, Kdm5a) was detected. All studied H9M-ciMPs demonstrated adaptation to T-lymphoid microenvironmental conditions while maintaining immature myelomonocytic features. Thus, the established system is relevant to leukaemia and stem cell biology.

scholarly journals Illuminating the cells: transient transformation of citrus to study gene functions and organelle activities related to fruit quality

2021 ◽  
Vol 8 (1) ◽  
Author(s):  
Jinli Gong ◽  
Zhen Tian ◽  
Xiaolu Qu ◽  
Qiunan Meng ◽  
Yajie Guan ◽  
...  
Keyword(s):  
Membrane Trafficking ◽  
Protein Localization ◽  
Cell Physiology ◽  
Transient Transformation ◽  
Citrus Fruits ◽  
Efficient System ◽  
Fluorescent Reporters ◽  
Biology Research ◽  
Insight Into ◽  
Microscopic Techniques

AbstractAlthough multiple microscopic techniques have been applied to horticultural research, few studies of individual organelles in living fruit cells have been reported to date. In this paper, we established an efficient system for the transient transformation of citrus fruits using an Agrobacterium-mediated method. Kumquat (Fortunella crassifolia Swingle) was used; it exhibits higher transformation efficiency than all citrus fruits that have been tested and a prolonged-expression window. Fruits were transformed with fluorescent reporters, and confocal microscopy and live-cell imaging were used to study their localization and dynamics. Moreover, various pH sensors targeting different subcellular compartments were expressed, and the local pH environments in cells from different plant tissues were compared. The results indicated that vacuoles are most likely the main organelles that contribute to the low pH of citrus fruits. In summary, our method is effective for studying various membrane trafficking events, protein localization, and cell physiology in fruit and can provide new insight into fruit biology research.

scholarly journals A cellular endolysosome-modulating pore-forming protein from a toad is negatively regulated by its paralog under oxidizing conditions

2020 ◽  
Vol 295 (30) ◽  
pp. 10293-10306 ◽  
Author(s):  
Qiquan Wang ◽  
Xianling Bian ◽  
Lin Zeng ◽  
Fei Pan ◽  
Lingzhen Liu ◽  
...  
Keyword(s):  
Disulfide Bond ◽  
Cell Biology ◽  
Biochemical Properties ◽  
Bacterial Toxin ◽  
Cell Physiology ◽  
Dependent Manner ◽  
Membrane Pore ◽  
Intracellular Vesicles

Endolysosomes are key players in cell physiology, including molecular exchange, immunity, and environmental adaptation. They are the molecular targets of some pore-forming aerolysin-like proteins (ALPs) that are widely distributed in animals and plants and are functionally related to bacterial toxin aerolysins. βγ-CAT is a complex of an ALP (BmALP1) and a trefoil factor (BmTFF3) in the firebelly toad (Bombina maxima). It is the first example of a secreted endogenous pore-forming protein that modulates the biochemical properties of endolysosomes by inducing pore formation in these intracellular vesicles. Here, using a large array of biochemical and cell biology methods, we report the identification of BmALP3, a paralog of BmALP1 that lacks membrane pore-forming capacity. We noted that both BmALP3 and BmALP1 contain a conserved cysteine in their C-terminal regions. BmALP3 was readily oxidized to a disulfide bond-linked homodimer, and this homodimer then oxidized BmALP1 via disulfide bond exchange, resulting in the dissociation of βγ-CAT subunits and the elimination of biological activity. Consistent with its behavior in vitro, BmALP3 sensed environmental oxygen tension in vivo, leading to modulation of βγ-CAT activity. Interestingly, we found that this C-terminal cysteine site is well conserved in numerous vertebrate ALPs. These findings uncover the existence of a regulatory ALP (BmALP3) that modulates the activity of an active ALP (BmALP1) in a redox-dependent manner, a property that differs from those of bacterial toxin aerolysins.

scholarly journals Mapping the phenotypic repertoire of the cytoplasmic 2-Cys peroxiredoxin – thioredoxin system. 1. Understanding commonalities and differences among cell types

2017 ◽  
Author(s):  
Gianluca Selvaggio ◽  
Pedro M. B. M. Coelho ◽  
Armindo Salvador
Keyword(s):  
Cell Lines ◽  
Thioredoxin Reductase ◽  
Cell Types ◽  
Cell Physiology ◽  
Related Factor ◽  
Similar Response ◽  
Redox Biology ◽  
Analytical Approximations ◽  
Thioredoxin System ◽  
Reduction Capacity

AbstractThe system (PTTRS) formed by typical 2-Cys peroxiredoxins (Prx), thioredoxin (Trx), Trx reductase (TrxR), and sulfiredoxin (Srx) is central in antioxidant protection and redox signaling in the cytoplasm of eukaryotic cells. Understanding how the PTTRS integrates these functions requires tracing phenotypes to molecular properties, which is non-trivial. Here we analyze this problem based on a model that captures the PTTRS’ conserved features. We have mapped the conditions that generate each distinct response to H2O2 supply rates (νsup), and estimated the parameters for thirteen human cell types and for Saccharomyces cerevisiae. The resulting composition-to-phenotype map yielded the following experimentally testable predictions. The PTTRS permits many distinct responses including ultra-sensitivity and hysteresis. However, nearly all tumor cell lines showed a similar response characterized by limited Trx-S- depletion and a substantial but self-limited gradual accumulation of hyperoxidized Prx at high νsup. This similarity ensues from strong correlations between the TrxR, Srx and Prx activities over cell lines, which contribute to maintain the Prx-SS reduction capacity in slight excess over the maximal steady state Prx-SS production. In turn, in erythrocytes, hepatocytes and HepG2 cells high νsup depletes Trx-S- and oxidizes Prx mainly to Prx-SS. In all nucleated human cells the Prx-SS reduction capacity defined a threshold separating two different regimes. At sub-threshold νsup cytoplasmic H2O2 is determined by Prx, nM-range and spatially localized, whereas at supra-threshold νsup it is determined by much less active alternative sinks and μM-range throughout the cytoplasm. The yeast shows a distinct response where the Prx Tsa1 accumulates in sulfenate form at high νsup. This is mainly due to an exceptional stability of Tsa1’s sulfenate.The implications of these findings for thiol redox regulation and cell physiology are discussed. All estimates were thoroughly documented and provided, together with analytical approximations for system properties, as a resource for quantitative redox biology.AbbreviationsASK1, apoptosis signal-regulating kinase 1; Cat, catalase; GSH, glutathione; GPx1, glutathione peroxidase 1; Grx, glutaredoxin; KEAP1, Kelch-like ECH-associated protein 1; NRF2, nuclear factor erythroid 2-related factor 2; Prx, typical 2-Cys peroxiredoxin; PTTRS, peroxiredoxin / thioredoxin / thioredoxin reductase system; Srx, sulfiredoxin; Trx, thioredoxin; TrxR, thioredoxin reductase.

scholarly journals Hemodynamic forces can be accurately measured in vivo with optical tweezers

2017 ◽  
Author(s):  
Sébastien Harlepp ◽  
Fabrice Thalmann ◽  
Gautier Follain ◽  
Jacky G. Goetz
Keyword(s):  
Optical Tweezers ◽  
Cell Biology ◽  
Accurate Determination ◽  
Force Measurements ◽  
Hemodynamic Forces ◽  
Non Invasive ◽  
Drag Forces ◽  
Measured Flow ◽  
Living Organisms

AbstractForce sensing and generation at the tissular and cellular scale is central to many biological events. There is a growing interest in modern cell biology for methods enabling force measurements in vivo. Optical trapping allows non-invasive probing of pico-Newton forces and thus emerged as a promising mean for assessing biomechanics in vivo. Nevertheless, the main obstacles rely in the accurate determination of the trap stiffness in heterogeneous living organisms, at any position where the trap is used. A proper calibration of the trap stiffness is thus required for performing accurate and reliable force measurements in vivo. Here, we introduce a method that overcomes these difficulties by accurately measuring hemodynamic profiles in order to calibrate the trap stiffness. Doing so, and using numerical methods to assess the accuracy of the experimental data, we measured flow profiles and drag forces imposed to trapped red blood cells of living zebrafish embryos. Using treatments enabling blood flow tuning, we demonstrated that such method is powerful in measuring hemodynamic forces in vivo with accuracy and confidence. Altogether, this study demonstrates the power of optical tweezing in measuring low range hemodynamic forces in vivo and offers an unprecedented tool in both cell and developmental biology.

scholarly journals Eukaryotic systems broaden the scope of synthetic biology

2009 ◽  
Vol 187 (5) ◽  
pp. 589-596 ◽  
Author(s):  
Karmella A. Haynes ◽  
Pamela A. Silver
Keyword(s):  
Synthetic Biology ◽  
Nucleic Acids ◽  
Cell Biology ◽  
Cell Behavior ◽  
Cellular Functions ◽  
Complex Cells ◽  
Cellular Processes ◽  
Challenges And Opportunities ◽  
Foundational Work ◽  
Biology Research

Synthetic biology aims to engineer novel cellular functions by assembling well-characterized molecular parts (i.e., nucleic acids and proteins) into biological “devices” that exhibit predictable behavior. Recently, efforts in eukaryotic synthetic biology have sprung from foundational work in bacteria. Designing synthetic circuits to operate reliably in the context of differentiating and morphologically complex cells presents unique challenges and opportunities for progress in the field. This review surveys recent advances in eukaryotic synthetic biology and describes how synthetic systems can be linked to natural cellular processes in order to manipulate cell behavior and to foster new discoveries in cell biology research.

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