Morphological Observation of the Cashmere Goat Fetal Fibroblasts after mTOR Kinase Inhibition with Combination of Fluorescent Dyes and Confocal Cell Imaging

2011 ◽  
Vol 343-344 ◽  
pp. 590-593
Author(s):  
Yan Liang ◽  
Xiao Jing Wang ◽  
Shu Yu Li ◽  
Cheberi ◽  
Zhi Gang Wang ◽  
...  
Keyword(s):  
Inner Mongolia ◽  
Mammalian Cells ◽  
Fluorescent Dyes ◽  
Cell Structure ◽  
Critical Role ◽  
Laser Scanning Microscopy ◽  
Morphological Observation ◽  
Cashmere Goat ◽  
Mtor Kinase ◽  
Fetal Fibroblasts

The mammalian target of rapamycin（mTOR）is a kind of Ser/Thr kinase in mammalian cells. It can recruit and integrate input signals from nutrients, growth factors, energy and environmental stress to regulate cell growth and proliferation via different cellular processes. This study uses the fetal fibroblasts of Inner Mongolia Cashmere goat (Capra hircas) to prove that the mTOR plays a critical role in formation of the cell cytoskeleton structure. The mTOR kinase activity was inhibited in Inner Mongolia Cashmere goat fetal fibroblasts (GFb) after treatment with CCI-779 (temsirolimus), an mTOR specific inhibitor for 48 h. The results showed that GFb cells were sensitive to CCI-779. GFb cells morphology and its cytoskeleton structure changed under confocal laser scanning microscopy stained with the Fluorescent phalloidin (50µg/ml, Phalloidin-FITC5282) which combines with F-actin. In summary, mTOR signaling pathway was proved to be functional in GFb cells and acts as a key regulator to form cell structure. The Morphological results indicated that perhaps the synthesis of microfilament or organization of cytoskeleton was disrupted in GFb cells when mTOR was inhibited.

Vacuoles Induced in Mammalian Cells by Helicobacter Cytotoxin are Acidic Organelles

1990 ◽  
Vol 48 (3) ◽  
pp. 168-169
Author(s):  
M. H. Chestnut ◽  
C. E. Catrenich
Keyword(s):  
Acridine Orange ◽  
Mammalian Cells ◽  
Laser Scanning ◽  
Laser Scanning Microscopy ◽  
Confocal Laser ◽  
Ulcer Disease ◽  
Gastroduodenal Disease ◽  
H Pylori

Helicobacter pylori is a non-invasive, Gram-negative spiral bacterium first identified in 1983, and subsequently implicated in the pathogenesis of gastroduodenal disease including gastritis and peptic ulcer disease. Cytotoxic activity, manifested by intracytoplasmic vacuolation of mammalian cells in vitro, was identified in 55% of H. pylori strains examined. The vacuoles increase in number and size during extended incubation, resulting in vacuolar and cellular degeneration after 24 h to 48 h. Vacuolation of gastric epithelial cells is also observed in vivo during infection by H. pylori. A high molecular weight, heat labile protein is believed to be responsible for vacuolation and to significantly contribute to the development of gastroduodenal disease in humans. The mechanism by which the cytotoxin exerts its effect is unknown, as is the intracellular origin of the vacuolar membrane and contents. Acridine orange is a membrane-permeant weak base that initially accumulates in low-pH compartments. We have used acridine orange accumulation in conjunction with confocal laser scanning microscopy of toxin-treated cells to begin probing the nature and origin of these vacuoles.

Autophagy Modulators and Neuroinflammation

2020 ◽  
Vol 27 (6) ◽  
pp. 955-982 ◽  
Author(s):  
Kyoung Sang Cho ◽  
Jang Ho Lee ◽  
Jeiwon Cho ◽  
Guang-Ho Cha ◽  
Gyun Jee Song
Keyword(s):  
Neurodegenerative Disorders ◽  
Neurological Disorders ◽  
Mammalian Cells ◽  
Critical Role ◽  
Therapeutic Agents ◽  
Mechanisms Of Action ◽  
Scientific Interest ◽  
Therapeutic Tools ◽  
Underlying Mechanisms

Background: Neuroinflammation plays a critical role in the development and progression of various neurological disorders. Therefore, various studies have focused on the development of neuroinflammation inhibitors as potential therapeutic tools. Recently, the involvement of autophagy in the regulation of neuroinflammation has drawn substantial scientific interest, and a growing number of studies support the role of impaired autophagy in the pathogenesis of common neurodegenerative disorders. Objective: The purpose of this article is to review recent research on the role of autophagy in controlling neuroinflammation. We focus on studies employing both mammalian cells and animal models to evaluate the ability of different autophagic modulators to regulate neuroinflammation. Methods: We have mostly reviewed recent studies reporting anti-neuroinflammatory properties of autophagy. We also briefly discussed a few studies showing that autophagy modulators activate neuroinflammation in certain conditions. Results: Recent studies report neuroprotective as well as anti-neuroinflammatory effects of autophagic modulators. We discuss the possible underlying mechanisms of action of these drugs and their potential limitations as therapeutic agents against neurological disorders. Conclusion: Autophagy activators are promising compounds for the treatment of neurological disorders involving neuroinflammation.

scholarly journals PGC-1-Related Coactivator, a Novel, Serum-Inducible Coactivator of Nuclear Respiratory Factor 1-Dependent Transcription in Mammalian Cells

2001 ◽  
Vol 21 (11) ◽  
pp. 3738-3749 ◽  
Author(s):  
Ulf Andersson ◽  
Richard C. Scarpulla
Keyword(s):  
Transcriptional Activation ◽  
Mammalian Cells ◽  
Laser Scanning ◽  
Laser Scanning Microscopy ◽  
Confocal Laser ◽  
Peroxisome Proliferator ◽  
Nuclear Respiratory Factor ◽  
Nuclear Respiratory Factor 1 ◽  
Cell Extracts

ABSTRACT The thermogenic peroxisome proliferator-activated receptor γ (PPAR-γ) coactivator 1 (PGC-1) has previously been shown to activate mitochondrial biogenesis in part through a direct interaction with nuclear respiratory factor 1 (NRF-1). In order to identify related coactivators that act through NRF-1, we searched the databases for sequences with similarities to PGC-1. Here, we describe the first characterization of a 177-kDa transcriptional coactivator, designated PGC-1-related coactivator (PRC). PRC is ubiquitously expressed in murine and human tissues and cell lines; but unlike PGC-1, PRC was not dramatically up-regulated during thermogenesis in brown fat. However, its expression was down-regulated in quiescent BALB/3T3 cells and was rapidly induced by reintroduction of serum, conditions where PGC-1 was not detected. PRC activated NRF-1-dependent promoters in a manner similar to that observed for PGC-1. Moreover, NRF-1 was immunoprecipitated from cell extracts by antibodies directed against PRC, and both proteins were colocalized to the nucleoplasm by confocal laser scanning microscopy. PRC interacts in vitro with the NRF-1 DNA binding domain through two distinct recognition motifs that are separated by an unstructured proline-rich region. PRC also contains a potent transcriptional activation domain in its amino terminus adjacent to an LXXLL motif. The spatial arrangement of these functional domains coincides with those found in PGC-1, supporting the conclusion that PRC and PGC-1 are structurally and functionally related. We conclude that PRC is a functional relative of PGC-1 that operates through NRF-1 and possibly other activators in response to proliferative signals.

scholarly journals RanBP2 and SENP3 Function in a Mitotic SUMO2/3 Conjugation-Deconjugation Cycle on Borealin

2009 ◽  
Vol 20 (1) ◽  
pp. 410-418 ◽  
Author(s):  
Ulf R. Klein ◽  
Markus Haindl ◽  
Erich A. Nigg ◽  
Stefan Muller
Keyword(s):  
Mammalian Cells ◽  
Spindle Assembly Checkpoint ◽  
Critical Role ◽  
Specific Interaction ◽  
Regulatory Function ◽  
Mitotic Progression ◽  
Chromosome Congression ◽  
Chromosomal Passenger

The ubiquitin-like SUMO system controls cellular key functions, and several lines of evidence point to a critical role of SUMO for mitotic progression. However, in mammalian cells mitotic substrates of sumoylation and the regulatory components involved are not well defined. Here, we identify Borealin, a component of the chromosomal passenger complex (CPC), as a mitotic target of SUMO. The CPC, which additionally comprises INCENP, Survivin, and Aurora B, regulates key mitotic events, including chromosome congression, the spindle assembly checkpoint, and cytokinesis. We show that Borealin is preferentially modified by SUMO2/3 and demonstrate that the modification is dynamically regulated during mitotic progression, peaking in early mitosis. Intriguingly, the SUMO ligase RanBP2 interacts with the CPC, stimulates SUMO modification of Borealin in vitro, and is required for its modification in vivo. Moreover, the SUMO isopeptidase SENP3 is a specific interaction partner of Borealin and catalyzes the removal of SUMO2/3 from Borealin. These data thus delineate a mitotic SUMO2/3 conjugation–deconjugation cycle of Borealin and further assign a regulatory function of RanBP2 and SENP3 in the mitotic SUMO pathway.

Cytokinesis arrest and redistribution of actin-cytoskeleton regulatory components in cells expressing the Rho GTPase CDC42Hs

1996 ◽  
Vol 109 (2) ◽  
pp. 367-377 ◽  
Author(s):  
H. Dutartre ◽  
J. Davoust ◽  
J.P. Gorvel ◽  
P. Chavrier
Keyword(s):  
Actin Cytoskeleton ◽  
Rho Gtpases ◽  
Mammalian Cells ◽  
Laser Scanning ◽  
Rho Gtpase ◽  
Laser Scanning Microscopy ◽  
Regulatory Subunit ◽  
Confocal Laser ◽  
Complex Architectures ◽  
Immunofluorescence Labelling

In mammalian cells, Rho GTPases control the reorganisation of the actin cytoskeleton in response to growth factors. In the cytoplasm, the polymerisation of actin filaments and their organisation into complex architectures is orchestrated by numerous proteins which act either directly, by interacting with actin, or by producing secondary messengers which serve as mediators between signal transduction pathways and the microfilament organisation. We sought to determine whether the intracellular distribution of some of these regulatory components may be controlled by the Rho GTPase CDC42Hs. With this aim, we have established HeLa-derived human cell lines in which expression of a constitutively activated mutant of CDC42Hs is inducible. Morphological analysis by immunofluorescence labelling and confocal laser scanning microscopy revealed a massive reorganisation of F-actin in cortical microspikes as well as podosome-like structures located at the ventral face of the cells. Concomitantly, the cells became giant and multinucleate indicating that cytokinesis was impaired. The actin bundling protein T-plastin, the vasodilatator-stimulated phosphoprotein (VASP), a profilin ligand, as well as the 85 kDa regulatory subunit of the phosphoinosite 3-kinase redistributed with F-actin into the CDC42Hs-induced structures.

scholarly journals E2F activity is regulated by cell cycle-dependent changes in subcellular localization.

1997 ◽  
Vol 17 (12) ◽  
pp. 7268-7282 ◽  
Author(s):  
R Verona ◽  
K Moberg ◽  
S Estes ◽  
M Starz ◽  
J P Vernon ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Subcellular Localization ◽  
Nuclear Localization ◽  
Mammalian Cells ◽  
Critical Role ◽  
Biological Properties ◽  
Dependent Manner ◽  
Restriction Point ◽  
Cycle Dependent ◽  
Almost All

E2F directs the cell cycle-dependent expression of genes that induce or regulate the cell division process. In mammalian cells, this transcriptional activity arises from the combined properties of multiple E2F-DP heterodimers. In this study, we show that the transcriptional potential of individual E2F species is dependent upon their nuclear localization. This is a constitutive property of E2F-1, -2, and -3, whereas the nuclear localization of E2F-4 is dependent upon its association with other nuclear factors. We previously showed that E2F-4 accounts for the majority of endogenous E2F species. We now show that the subcellular localization of E2F-4 is regulated in a cell cycle-dependent manner that results in the differential compartmentalization of the various E2F complexes. Consequently, in cycling cells, the majority of the p107-E2F, p130-E2F, and free E2F complexes remain in the cytoplasm. In contrast, almost all of the nuclear E2F activity is generated by pRB-E2F. This complex is present at high levels during G1 but disappears once the cells have passed the restriction point. Surprisingly, dissociation of this complex causes little increase in the levels of nuclear free E2F activity. This observation suggests that the repressive properties of the pRB-E2F complex play a critical role in establishing the temporal regulation of E2F-responsive genes. How the differential subcellular localization of pRB, p107, and p130 contributes to their different biological properties is also discussed.

scholarly journals Membrane permeabilization of mammalian cells using bursts of high magnetic field pulses

PeerJ ◽  
2017 ◽  
Vol 5 ◽  
pp. e3267 ◽  
Author(s):  
Vitalij Novickij ◽  
Janja Dermol ◽  
Audrius Grainys ◽  
Matej Kranjc ◽  
Damijan Miklavčič
Keyword(s):  
Mammalian Cells ◽  
Fluorescent Dyes ◽  
Pulse Generator ◽  
Chinese Hamster ◽  
Electrical Conductance ◽  
Single Pulse ◽  
Membrane Permeabilization ◽  
Contactless Method ◽  
Dependent Manner ◽  
Cell Permeabilization

Background Cell membrane permeabilization by pulsed electromagnetic fields (PEMF) is a novel contactless method which results in effects similar to conventional electroporation. The non-invasiveness of the methodology, independence from the biological object homogeneity and electrical conductance introduce high flexibility and potential applicability of the PEMF in biomedicine, food processing, and biotechnology. The inferior effectiveness of the PEMF permeabilization compared to standard electroporation and the lack of clear description of the induced transmembrane transport are currently of major concern. Methods The PEMF permeabilization experiments have been performed using a 5.5 T, 1.2 J pulse generator with a multilayer inductor as an applicator. We investigated the feasibility to increase membrane permeability of Chinese Hamster Ovary (CHO) cells using short microsecond (15 µs) pulse bursts (100 or 200 pulses) at low frequency (1 Hz) and high dB/dt (>106 T/s). The effectiveness of the treatment was evaluated by fluorescence microscopy and flow cytometry using two different fluorescent dyes: propidium iodide (PI) and YO-PRO®-1 (YP). The results were compared to conventional electroporation (single pulse, 1.2 kV/cm, 100 µs), i.e., positive control. Results The proposed PEMF protocols (both for 100 and 200 pulses) resulted in increased number of permeable cells (70 ± 11% for PI and 67 ± 9% for YP). Both cell permeabilization assays also showed a significant (8 ± 2% for PI and 35 ± 14% for YP) increase in fluorescence intensity indicating membrane permeabilization. The survival was not affected. Discussion The obtained results demonstrate the potential of PEMF as a contactless treatment for achieving reversible permeabilization of biological cells. Similar to electroporation, the PEMF permeabilization efficacy is influenced by pulse parameters in a dose-dependent manner.

scholarly journals Low Concentrations of Chlorhexidine Inhibit the Formation and Structural Integrity of Enzyme-Treated Multispecies Oral Biofilms

2021 ◽  
Vol 12 ◽  
Author(s):  
Kay Andrin Gränicher ◽  
Lamprini Karygianni ◽  
Thomas Attin ◽  
Thomas Thurnheer
Keyword(s):  
Structural Integrity ◽  
Fluorescent Dyes ◽  
Combined Treatment ◽  
Extracellular Proteins ◽  
Laser Scanning Microscopy ◽  
Extracellular Dna ◽  
Anaerobic Growth ◽  
Low Concentration ◽  
Low Concentrations ◽  
Dental Hard Tissues

The self-produced matrix of biofilms, consisting of extracellular polymeric substances, plays an important role in biofilm adhesion to surfaces and the structural integrity of biofilms. In dentistry, biofilms cause multiple diseases such as caries, periodontitis, and pulpitis. Disruption of these biofilms adhering to dental hard tissues may pose a major challenge since biofilms show higher tolerance to antimicrobials and antibiotics than planktonic cells. In this study, the effect of low concentrations of chlorhexidine (CHX) on enzyme-treated multispecies oral biofilm was investigated in an in vitro model. Six-species biofilms were enzymatically treated by anaerobic growth in a medium containing DNase I and proteinase K. Biofilms were exposed to a low concentration of CHX at defined time points. After 64h, biofilms were either harvested and quantified by cultural analyses or stained for confocal laser scanning microscopy (CLSM) analyses using either Live/Dead kit or different fluorescent dyes. A mixture of YoPro1 and SYTOX™ Green, Fluorescent Brightener 28 (Calcofluor), and SYPRO™ Ruby Protein Gel Stain was used to stain total DNA, exopolysaccharides, and extracellular proteins, respectively. Extracellular DNA (eDNA) was visualized via an indirect immunofluorescence assay (Mouse anti-DNA IgG, Goat anti-Mouse IgG, Streptavidin-Cy3). Overall, the total colony-forming units significantly decreased after combined treatment with a low concentration of CHX and enzymes compared to the group treated with CHX alone (p<0.001). These findings also apply to five species individually (Streptococcus mutans, Streptococcus oralis, Actinomyces oris, Veillonella dispar, and Candida albicans) occurring in the biofilms, with Fusobacterium nucleatum being the only exception. Furthermore, CLSM images showed less dense biofilms and a reduction in cell numbers after combined treatment compared to the group without enzymes. The combination of enzymes capable of disturbing the matrix integrity with antimicrobial agents thus appears to be a promising approach for biofilm disruption and killing.

Oxygen sensing and molecular adaptation to hypoxia

1996 ◽  
Vol 76 (3) ◽  
pp. 839-885 ◽  
Author(s):  
H. F. Bunn ◽  
R. O. Poyton
Keyword(s):  
Transcription Factors ◽  
Mammalian Cells ◽  
Critical Role ◽  
Hypoxia Inducible Factor ◽  
Signal Transduction Pathway ◽  
Adaptation To Hypoxia ◽  
Adaptive Responses ◽  
Expression Of Genes ◽  
Activation Of Transcription ◽  
Activation Of Oxygen

This review focuses on the molecular stratagems utilized by bacteria, yeast, and mammals in their adaptation to hypoxia. Among this broad range of organisms, changes in oxygen tension appear to be sensed by heme proteins, with subsequent transfer of electrons along a signal transduction pathway which may depend on reactive oxygen species. These heme-based sensors are generally two-domain proteins. Some are hemokinases, while others are flavohemoproteins [flavohemoglobins and NAD(P)H oxidases]. Hypoxia-dependent kinase activation of transcription factors in nitrogen-fixing bacteria bears a striking analogy to the phosphorylation of hypoxia inducible factor-1 (HIF-1) in mammalian cells. Moreover, redox chemistry appears to play a critical role both in the trans-activation of oxygen-responsive genes in unicellular organisms as well as in the activation of HIF-1. In yeast and bacteria, regulatory operons coordinate expression of genes responsible for adaptive responses to hypoxia and hyperoxia. Similarly, in mammals, combinatorial interactions of HIF-1 with other identified transcription factors are required for the hypoxic induction of physiologically important genes.

Quantification of microtubule dynamics in living plant cells using fluorescence redistribution after photobleaching

1994 ◽  
Vol 107 (4) ◽  
pp. 775-784 ◽  
Author(s):  
J.M. Hush ◽  
P. Wadsworth ◽  
D.A. Callaham ◽  
P.K. Hepler
Keyword(s):  
Mammalian Cells ◽  
Laser Scanning ◽  
Dynamic Instability ◽  
Animal Cell ◽  
Plant Cells ◽  
Preprophase Band ◽  
Laser Scanning Microscopy ◽  
Confocal Laser ◽  
Turnover Rates ◽  
Living Plant

Microtubule (MT) turnover within the four principal MT arrays, the cortical array, the preprophase band, the mitotic spindle and the phragmoplast, has been measured in living stamen hair cells of Tradescantia that have been injected with fluorescent neurotubulin. Using the combined techniques of confocal laser scanning microscopy and fluorescence redistribution after photobleaching (FRAP), we report that the half-time of turnover in spindle MTs is t 1/2 = 31 +/- 6 seconds, which is in excellent agreement with previous measurements of turnover in animal cell spindles. Tradescantia interphase MTs, however, exhibit turnover rates (t 1/2 = 67 +/- seconds) that are some 3.4-fold faster than those measured in interphase mammalian cells, and thus are revealed as being highly dynamic. Preprophase band and phragmoplast MTs have turnover rates similar to those of interphase MTs in Tradescantia. The spatial and temporal aspects of the fluorescence redistribution after photobleaching in all four MT arrays are more consistent with subunit exchange by the mechanism of dynamic instability than treadmilling. This is the first quantification of MT dynamics in plant cells.

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