scholarly journals Inhibition of Suicidal Erythrocyte Death by Indirubin-3’-Monoxime

2018 ◽  
Vol 45 (3) ◽  
pp. 1108-1120 ◽  
Author(s):  
Chunqiu Liu ◽  
Peipei Jiang ◽  
Yuanhong Xu ◽  
Meijuan Zheng ◽  
Jinpin Qiao ◽  
...  
Keyword(s):  
Laser Scanning ◽  
Human Tumor ◽  
Annexin V ◽  
Fluorescein Isothiocyanate ◽  
Human Erythrocytes ◽  
Laser Scanning Microscopy ◽  
Confocal Laser ◽  
Forward Scatter ◽  
Energy Depletion ◽  
Ceramide Level

Background/Aims: Qing Dai is a prized traditional Chinese medicine whose major component, indirubin, and its derivative, indirubin-3’-monoxime (IDM), have inhibitory effects on the growth of many human tumor cells and pronounced anti-leukemic activities. However, the effects of IDM on mature human erythrocytes are unclear. This study aimed to evaluate the potential impact of IDM on erythrocytes and the mechanisms underlying that impact. Methods: Utilizing flow cytometry and confocal laser scanning microscopy, phosphatidylserine exposure at the cell surface was estimated by annexin V-fluorescein isothiocyanate (FITC). The relative cell size, expressed in arbitrary units, was evaluated by forward scatter in a flow cytometer. Fluo-3 fluorescence was used to bewrite changes in cytosolic Ca2+ activity, reactive oxygen species (ROS) formation was assessed by 2,7-dichlorodihydrofluorescein diacetate (DCFH-DA) fluorescence, and ceramide abundance was evaluated by FITC-conjugated specific antibodies. Results: The 24-h exposure of human erythrocytes to IDM (12 µM) significantly decreased the percentage of annexin V-binding erythrocytes and the intracellular calcium concentration ([Ca2+]i). IDM (3-12 µM) did not significantly modify the ceramide level or DCFH-DA fluorescence. Energy depletion (removal of glucose for 24 hours) significantly increased annexin V binding and Fluo-3 fluorescence and diminished forward scatter, and these effects were significantly mitigated by IDM (12 µM). Moreover, the Ca2+ ionophore ionomycin (1 µM, 60 min) and oxidative stress (30 min exposure to 0.05 mM tert-butyl hydroperoxide, t-BHP) similarly triggered eryptosis, which was also significantly suppressed by IDM. Conclusions: IDM is a novel inhibitor of suicidal erythrocyte death following ionomycin treatment, t-BHP treatment and energy depletion. Thus, IDM may counteract anemia and impairment of microcirculation, at least in part, by inhibition of Ca2+ entry into erythrocytes.

scholarly journals Primary Neurons and Differentiated NSC-34 Cells Are More Susceptible to Arginine-Rich ALS Dipeptide Repeat Protein-Associated Toxicity than Non-Differentiated NSC-34 and CHO Cells

2019 ◽  
Vol 20 (24) ◽  
pp. 6238 ◽  
Author(s):  
Anna L. Gill ◽  
Monica Z. Wang ◽  
Beth Levine ◽  
Alan Premasiri ◽  
Fernando G. Vieira
Keyword(s):  
Motor Neurons ◽  
Laser Scanning ◽  
Neuronal Cell ◽  
Fluorescein Isothiocyanate ◽  
Laser Scanning Microscopy ◽  
Confocal Laser ◽  
Primary Neurons ◽  
Repeat Protein ◽  
Neuronal Cell Lines ◽  
Dipeptide Repeat

A repeat expansion mutation in the C9orf72 gene is the most common known genetic cause of amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). In this study, using multiple cell-based assay systems, we reveal both increased dipeptide repeat protein (DRP) toxicity in primary neurons and in differentiated neuronal cell lines. Using flow cytometry and confocal laser scanning microscopy of cells treated with fluorescein isothiocyanate (FITC)-labeled DRPs, we confirm that poly-glycine-arginine (GR) and poly-proline-arginine (PR) DRPs entered cells more readily than poly-glycine-proline (GP) and poly-proline-alanine (PA) DRPs. Our findings suggest that the toxicity of C9-DRPs may be influenced by properties associated with differentiated and aging motor neurons. Further, our findings provide sensitive cell-based assay systems to test phenotypic rescue ability of potential interventions.

scholarly journals Macropinocytosis as a Mechanism of Entry into Primary Human Urethral Epithelial Cells by Neisseria gonorrhoeae

2000 ◽  
Vol 68 (3) ◽  
pp. 1696-1699 ◽  
Author(s):  
Michael K. Zenni ◽  
Peter C. Giardina ◽  
Hillery A. Harvey ◽  
Jianqiang Shao ◽  
Margaret R. Ketterer ◽  
...  
Keyword(s):  
Epithelial Cells ◽  
Laser Scanning ◽  
Actin Polymerization ◽  
Kinase Inhibitors ◽  
Fluorescein Isothiocyanate ◽  
Laser Scanning Microscopy ◽  
Confocal Laser ◽  
Transmission Electron ◽  
Microscopy Analysis ◽  
Phosphoinositide 3 Kinase

ABSTRACT Gonococcal entry into primary human urethral epithelial cells (HUEC) can occur by macropinocytosis. Scanning and transmission electron microscopy revealed lamellipodia surrounding gonococci, and confocal laser scanning microscopy analysis showed organisms colocalized with M r 70,000 fluorescein isothiocyanate-labeled dextran within the cells. Phosphoinositide 3-kinase inhibitors and an actin polymerization inhibitor prevented macropinocytic entry of gonococci into HUEC.

Sudan Black B Reduces Autofluorescence in Murine Renal Tissue

2011 ◽  
Vol 135 (10) ◽  
pp. 1335-1342 ◽  
Author(s):  
Yan Sun ◽  
Hong Yu ◽  
Dong Zheng ◽  
Qi Cao ◽  
Ya Wang ◽  
...  
Keyword(s):  
Laser Scanning ◽  
Kidney Tissue ◽  
Fluorescein Isothiocyanate ◽  
Renal Tissue ◽  
Laser Scanning Microscopy ◽  
Confocal Laser ◽  
Frozen Sections ◽  
Paraffin Sections ◽  
Biopsy Material ◽  
Sudan Black B

Context.—Renal tissue emits intense autofluorescence, making it difficult to differentiate specific immunofluorescence signals and thus limiting its application to clinical biopsy material. Objective.—To identify and minimize autofluorescence of renal tissue and demonstrate a simple, efficient method to reduce autofluorescence using Sudan black B. Design.—In this study, the sources and features of autofluorescence emitted from kidney tissue were examined. Broad autofluorescence was visualized in both frozen and paraffin kidney sections of normal mice and mice with Adriamycin-induced nephropathy using confocal laser scanning microscopy. Autofluorescence appeared in commonly used 4′,6-diamidino-2-phenylindole, fluorescein isothiocyanate, and Texas Red channels but not in far-red channel, and emitted extensively from red cells, injured tubulointersitial cells, and protein casts in diseased kidney. To eliminate autofluorescence, Sudan black B was used on formaldehyde-fixed paraffin sections and frozen sections of mouse kidney. The effects of Sudan black B in various concentrations were tested on kidney tissue. Results.—The 0.1% Sudan black B effectively blocked autofluorescence from both paraffin and frozen sections without adversely affecting specific fluorescence signals. Interestingly, the solvent for Sudan black B, 70% ethanol, was also shown to reduce autofluorescence on frozen sections, but not on paraffin sections. Conclusions.—This study demonstrates a simple, efficient, and cost-effective method to reduce autofluorescence using Sudan black B, and also provides a comprehensive approach to identify and minimize autofluorescence of renal tissue.

scholarly journals Reconstructed three-dimensional images of flow-sorted nuclei hybridized with chromosome-specific probes.

1996 ◽  
Vol 44 (11) ◽  
pp. 1337-1343 ◽  
Author(s):  
M Matsuta ◽  
M Matsuta ◽  
S Hayashi ◽  
S Yasumi ◽  
K Sasaki ◽  
...  
Keyword(s):  
Laser Scanning ◽  
Three Dimensional ◽  
Fluorescein Isothiocyanate ◽  
S Phase ◽  
Laser Scanning Microscopy ◽  
Chromosome 17 ◽  
Confocal Laser ◽  
Cycle Phase ◽  
M Phase ◽  
Optimal Efficiency

We demonstrated that the three-dimensional (3-D) locational and morphological differences of chromosome 17 are dependent on each cell cycle phase in the clinical materials. Cell suspensions prepared from hypertrophied tonsil were hybridized with chromosome 17 whole painting probe or its centromeric probe and the probes were detected with fluorescein isothiocyanate. Then the cells were sorted from G(0+1), S-, and G(2+M)-phase fractions by flow cytometry and observed by confocal laser scanning microscopy to obtain the serial optical sections. The 3-D images were obtained by assembling these sections using a computerized image analysis device. The distribution of centromeric copies was analyzed statistically, and the data values were not a population of random distribution within a sphere. The copies were observed in the periphery of the nuclei in G(0+1)- and S-phase. The 3-D images revealed that chromosome 17 was oval in shape in the G(0+1)-phase nucleus, and was changing into a flame shape in the S-phase, with arms stretching out along the nuclear membrane, and looked bush shaped in G2-phase. The eccentric distribution of chromosome 17 in G(0+1)- and S-phase nuclei may reflect the optimal efficiency of incorporating and/or releasing essential materials and products.

Internalized SP-A and lipid are differentially resecreted by type II pneumocytes

2000 ◽  
Vol 278 (3) ◽  
pp. L580-L590 ◽  
Author(s):  
Heide Wissel ◽  
Stefan Zastrow ◽  
Ekkehard Richter ◽  
Paul A. Stevens
Keyword(s):  
Laser Scanning ◽  
Protein A ◽  
Fluorescein Isothiocyanate ◽  
Surfactant Protein ◽  
Laser Scanning Microscopy ◽  
Confocal Laser ◽  
Type Ii Cells ◽  
Type Ii ◽  
Type Ii Pneumocytes

Biochemical and morphological assays were developed to study surfactant protein A (SP-A) and lipid resecretion kinetics by isolated type II cells in vitro. After a 10-min uptake period with SP-A (3 μg/106 cells) in combination with liposomes (60 μg/106 cells), the cells were allowed to resecrete. After 5 min of resecretion, only 21.7 ± 4.6% of the internalized SP-A remained intracellularly compared with 54 ± 2.9% of the lipids. Extracellular SP-A present during the resecretion period partially inhibited resecretion (SP-A, 36% at 5 min; lipid, ∼16% at 5 min). Lipid resecretion was also dependent on the SP-A concentration present during the uptake period. Although, as shown by confocal laser scanning microscopy, after a 10-min uptake period at 37°C, most of the fluorescein isothiocyanate-labeled SP-A and rhodamine-phosphatidylethanolamine-labeled lipids colocalized within the cells, after an additional 10 min of resecretion, both the strength of the fluorescence signals and the extent of colocalization had markedly decreased. These data indicate that internalized lipid and SP-A can be resecreted rapidly by type II cells, likely via different pathways.

scholarly journals Ultrasmall gold and silver/gold nanoparticles (2 nm) as autofluorescent labels for poly(D,L-lactide-co-glycolide) nanoparticles (140 nm)

2020 ◽  
Vol 31 (12) ◽  
Author(s):  
Karolin Wey ◽  
Matthias Epple
Keyword(s):  
Gold Nanoparticles ◽  
Fluorescence Spectroscopy ◽  
Metallic Nanoparticles ◽  
Laser Scanning ◽  
Stokes Shift ◽  
Fluorescein Isothiocyanate ◽  
Plga Nanoparticles ◽  
Laser Scanning Microscopy ◽  
Confocal Laser ◽  
Water Emulsion

AbstractUltrasmall metallic nanoparticles show an efficient autofluorescence after excitation in the UV region, combined with a low degree of fluorescent bleaching. Thus, they can be used as fluorescent labels for polymer nanoparticles which are frequently used for drug delivery. A versatile water-in-oil-in-water emulsion-evaporation method was developed to load poly(D,L-lactide-co-glycolide) (PLGA) nanoparticles with autofluorescent ultrasmall gold and silver/gold nanoparticles (diameter 2 nm). The metallic nanoparticles were prepared by reduction of tetrachloroauric acid with sodium borohydride and colloidally stabilised with 11-mercaptoundecanoic acid. They were characterised by UV–Vis and fluorescence spectroscopy, showing a large Stokes shift of about 370 nm with excitation maxima at 250/270 nm and emission maxima at 620/640 nm for gold and silver/gold nanoparticles, respectively. The labelled PLGA nanoparticles (140 nm) were characterised by dynamic light scattering (DLS), scanning electron microscopy (SEM), and UV–Vis and fluorescence spectroscopy. Their uptake by HeLa cells was followed by confocal laser scanning microscopy. The metallic nanoparticles remained inside the PLGA particle after cellular uptake, demonstrating the efficient encapsulation and the applicability to label the polymer nanoparticle. In terms of fluorescence, the metallic nanoparticles were comparable to fluorescein isothiocyanate (FITC).

scholarly journals The Amelogenin-Derived Peptide TVH-19 Promotes Dentinal Tubule Occlusion and Mineralization

Polymers ◽  
2021 ◽  
Vol 13 (15) ◽  
pp. 2473
Author(s):  
Xiu Peng ◽  
Sili Han ◽  
Kun Wang ◽  
Longjiang Ding ◽  
Zhenqi Liu ◽  
...  
Keyword(s):  
Laser Scanning ◽  
Fluorescein Isothiocyanate ◽  
Laser Scanning Microscopy ◽  
Confocal Laser ◽  
Dentinal Tubule ◽  
X Ray ◽  
Electron Microscopy Analysis ◽  
Tubule Occlusion ◽  
Demineralized Dentin

In this study, the amelogenin-derived peptide, TVH-19, which has been confirmed to promote mineralization, was evaluated to derive its potential to induce dentinal tubule occlusion. The binding capability of fluorescein isothiocyanate (FITC)-labeled TVH-19 to the demineralized dentin surface was analyzed by confocal laser scanning microscopy (CLSM). Additionally, the sealing function of the peptide was studied through the remineralization of demineralized dentin in vitro. The adsorption results showed that TVH-19 could bind to the hydroxyapatite and demineralized dentin surfaces, especially to periodontal dentin. Scanning electron microscopy analysis further revealed that TVH-19 created mineral precipitates. The plugging rate in the TVH-19 group was higher than that in the PBS group. Moreover, energy-dispersive X-ray spectroscopy (EDX) results indicated that the calcium/phosphorus (Ca/P) ratio of the new minerals induced by TVH-19 was close to that of the hydroxyapatite. Attenuated total internal reflection-Fourier transform infrared (ATR-FTIR) spectrometry and X-ray diffraction (XRD) results indicated that the hydroxyapatite crystals formed via remineralization elongated the axial growth and closely resembled the natural dentin components. These findings indicate that TVH-19 can effectively promote dentin sealing by binding to the periodontal dentin, promoting mineral deposition, and reducing the space between the dentin tubules.

A megalin-like receptor is involved in protein endocytosis in the midgut of an insect (Bombyx mori, Lepidoptera)

2008 ◽  
Vol 295 (4) ◽  
pp. R1290-R1300 ◽  
Author(s):  
M. Casartelli ◽  
G. Cermenati ◽  
S. Rodighiero ◽  
F. Pennacchio ◽  
B. Giordana
Keyword(s):  
Bombyx Mori ◽  
Laser Scanning ◽  
Density Lipoprotein ◽  
Fluorescein Isothiocyanate ◽  
Laser Scanning Microscopy ◽  
Metabolic Inhibitors ◽  
Confocal Laser ◽  
Pcr Analysis ◽  
Apical Plasma Membrane ◽  
Microtubule Organization

The mechanism responsible for fluorescein isothiocyanate (FITC)-albumin internalization by columnar cells in culture obtained from the midgut of Bombyx mori larvae was examined by confocal laser scanning microscopy. Protein uptake changed over time, and it appeared to be energy dependent, since it was strongly reduced by both low temperatures and metabolic inhibitors. Labeled albumin uptake as a function of increasing protein concentration showed a saturation kinetics with a Michaelis constant value of 2.0 ± 0.6 μM. These data are compatible with the occurrence of receptor-mediated endocytosis. RT-PCR analysis and colocalization experiments with an anti-megalin primary antibody indicated that the receptor involved was a putative homolog of megalin, the multiligand endocytic receptor belonging to the low-density lipoprotein receptor family, responsible for the uptake of various molecules, albumin included, in many epithelial cells of mammals. This insect receptor, like the mammalian counterpart, required Ca2+ for albumin internalization and was inhibited by gentamicin. FITC-albumin internalization was clathrin mediated, since two inhibitors of this process caused a significant reduction of the uptake, and clathrin and albumin colocalized in the intermicrovillar areas of the apical plasma membrane. The integrity of actin and microtubule organization was essential for the correct functioning of the endocytic machinery.

scholarly journals Microscopic Photosensitization: A New Tool to Investigate the Role of Mitochondria in Cell Death

2002 ◽  
Vol 2 ◽  
pp. 1198-1208 ◽  
Author(s):  
May-Ghee Lum ◽  
Tetsuhiro Minamikawa ◽  
Phillip Nagley
Keyword(s):  
Cell Death ◽  
Laser Irradiation ◽  
Laser Scanning ◽  
Annexin V ◽  
Laser Scanning Microscopy ◽  
Confocal Laser ◽  
Rapid Loss ◽  
Active Involvement ◽  
Induction Of Apoptosis

Active involvement of mitochondria in cell death has been well-documented, but local apoptotic signaling between subsets of mitochondria has been poorly explored to date. Using mitochondrially localized CMXRos as a photosensitizer coupled to laser irradiation by confocal laser scanning microscopy, we demonstrate that partial irradiation of about half the mitochondria in human 143B TK–cells induces rapid loss of mitochondrial membrane potential (ΔΨm) in nonirradiated mitochondria. Cells so partially irradiated show apoptotic indications, including mobilization of cytochrome c and binding of annexin V within 2 h following irradiation. The loss of ΔΨm in nonirradiated mitochondria did not occur in cells photoirradiated in the absence of CMXRos. Increasing the proportion of irradiated mitochondria in each cell (up to about 50%) generated a correspondingly greater percentage of cells in which nonirradiated mitochondria lost ΔΨm and which also showed apoptotic indications. Only at the highest level of irradiation (global for all mitochondria in one cell) were signs of necrosis evident (judged by uptake of propidium iodide). Because laser irradiation is specific to the subpopulation of mitochondria targeted, the data imply that a signal emanating from irradiated mitochondria is processed by their nonirradiated counterparts. We conclude that intermitochondrial signaling occurs in the subcellular response to induction of apoptosis.

miR-21 Inhibits Cell Proliferation and Apoptosis Through Regulating the Phosphatase and Tensin Homolog/Phosphoinositide 3-Kinase/Akt Signaling Pathway in Cervical Cancer Cells

2020 ◽  
Vol 10 (2) ◽  
pp. 276-280
Author(s):  
Zhijia Zhan ◽  
Xiaoyan Xu ◽  
Zining Li ◽  
Xiujuan Chen ◽  
Qian Li ◽  
...  
Keyword(s):  
Cervical Cancer ◽  
Cancer Cells ◽  
Signaling Pathway ◽  
Cell Apoptosis ◽  
Laser Scanning ◽  
Annexin V ◽  
Akt Signaling ◽  
Laser Scanning Microscopy ◽  
Confocal Laser ◽  
Cervical Cancer Cells

A variety of miRNAs regulate cellular physiology and tumor cells. miR-21 regulates tumorigenesis though the modulating cell apoptosis, migration and invasiveness via activating PTEN/PI3K signaling pathway. This study aimed to assess miR-21’s effect on cervical cancer cells and the underlying mechanisms. HeLa were cultured in DMEM medium. Western blotting was to measure bcl-2, bax, p-Akt, PTEN protein level. Annexin V-FITC was measured to analyze the samples by using confocal laser scanning microscopy. Our result showed that miR-21 significantly induced the cell apoptosis of HeLa via decreasing the protein expression of bcl-2. miR-21 markedly inhibited PI3K/Akt signaling, increased PTEN level, induced cancer cell apoptosis and retarded the tumor angiogenesis and proliferation. miR-21 could regulate the proliferation of HeLa via upregulation of PTEN and downregulation of PI3K/Akt pathway, which provides a novel therapeutic insight for the therapy of cervical cancer.

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