Use of fluorescent dyes in the determination of adherence of human leucocytes to endothelial cells and the effect of fluorochromes on cellular function

The present knowledge of the three-dimensional structure of ribosomes is far too limited to enable a complete understanding of the various roles which ribosomes play in protein biosynthesis. The spatial arrangement of proteins and ribonuclec acids in ribosomes can be analysed in many ways. Determination of binding sites for individual proteins on ribonuclec acid and locations of the mutual positions of proteins on the ribosome using labeling with fluorescent dyes, cross-linking reagents, neutron-diffraction or antibodies against ribosomal proteins seem to be most successful approaches. Structure and function of ribosomes can be correlated be depleting the complete ribosomes of some proteins to the functionally inactive core and by subsequent partial reconstitution in order to regain active ribosomal particles.

Download Full-text

43. Determination of cryoprotectant permeability properties in monolayers of bovine endothelial cells using an in situ fluorescence quenching technique

Cryobiology ◽

10.1016/j.cryobiol.2009.10.057 ◽

2009 ◽

Vol 59 (3) ◽

pp. 382

Author(s):

A.K. Fry ◽

A.Z. Higgins

Keyword(s):

Endothelial Cells ◽

Fluorescence Quenching ◽

Bovine Endothelial Cells ◽

Fluorescence Quenching Technique

Download Full-text

Alpha-Asarone Protects Endothelial Cells from Injury by Angiotensin II

Evidence-based Complementary and Alternative Medicine ◽

10.1155/2014/682041 ◽

2014 ◽

Vol 2014 ◽

pp. 1-7 ◽

Cited By ~ 3

Author(s):

Hai-Xia Shi ◽

Jiajun Yang ◽

Tao Yang ◽

Yong-Liang Xue ◽

Jun Liu ◽

...

Keyword(s):

Nitric Oxide ◽

Endothelial Cells ◽

Angiotensin Ii ◽

Cell Injury ◽

Fluorescent Dyes ◽

Umbilical Vein ◽

Protective Effects ◽

Ang Ii ◽

Versus Model

α-Asarone is the major therapeutical constituent ofAcorus tatarinowiiSchott. In this study, the potential protective effects ofα-asarone against endothelial cell injury induced by angiotensin II were investigatedin vitro. The EA.hy926 cell line derived from human umbilical vein endothelial cells was pretreated withα-asarone (10, 50, 100 µmol/L) for 1 h, followed by coincubation with Ang II (0.1 µmol/L) for 24 h. Intracellular nitric oxide (NO) and reactive oxygen species (ROS) were detected by fluorescent dyes, and phosphorylation of endothelial nitric oxide synthase (eNOS) atSer1177was determined by Western blotting.α-Asarone dose-dependently mitigated the Ang II-induced intracellular NO reduction (P<0.01versus model) and ROS production (P<0.01versus model). Furthermore, eNOS phosphorylation (Ser1177) by acetylcholine was significantly inhibited by Ang II, while pretreatment for 1 h withα-asarone partially prevented this effect (P<0.05versus model). Additionally, cell viability determined by the MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] assay (105~114.5% versus control,P>0.05) was not affected after 24 h of incubation withα-asarone at 1–100 µmol/L. Therefore,α-asarone protects against Ang II-mediated damage of endothelial cells and may be developed to prevent injury to cardiovascular tissues.

Download Full-text

Accurate Determination of Human CPR Conformational Equilibrium by smFRET using Dual Orthogonal Non-Canonical Amino Acid Labeling

10.1101/407684 ◽

2018 ◽

Author(s):

Robert B. Quast ◽

Fataneh Fatemi ◽

Michel Kranendonk ◽

Emmanuel Margeat ◽

Gilles Truan

Keyword(s):

Single Molecule ◽

Conformational Equilibrium ◽

Fluorescent Dyes ◽

Photophysical Properties ◽

Accurate Determination ◽

Conformational Dynamics ◽

Resonance Energy Transfer ◽

Resonance Energy ◽

General Strategy

ABSTRACTConjugation of fluorescent dyes to proteins - a prerequisite for the study of conformational dynamics by single molecule Förster resonance energy transfer (smFRET) - can lead to substantial changes of the dye’s photophysical properties, ultimately biasing the quantitative determination of inter-dye distances. In particular the popular cyanine dyes and their derivatives, which are by far the most used dyes in smFRET experiments, exhibit such behavior. To overcome this, a general strategy to site-specifically equip proteins with FRET pairs by chemo-selective reactions using two distinct non-canonical amino acids simultaneously incorporated through genetic code expansion in Escherichia coli was developed. Applied to human NADPH- cytochrome P450 reductase (CPR), the importance of homogenously labeled samples for accurate determination of FRET efficiencies was demonstrated. Furthermore, the effect of NADP+ on the ionic strength dependent modulation of the conformational equilibrium of CPR was unveiled. Given its generality and accuracy, the presented methodology establishes a new benchmark to decipher complex molecular dynamics on single molecules.

Download Full-text

Pattern Analysis Meets Cell Biology

Microscopy and Microanalysis ◽

10.1017/s1431927600015877 ◽

1999 ◽

Vol 5 (S2) ◽

pp. 510-511

Author(s):

Robert F. Murphy ◽

Michael V. Boland

Keyword(s):

Cell Biology ◽

Fluorescent Dyes ◽

Protein A ◽

Chinese Hamster ◽

Subcellular Location ◽

Computer Applications ◽

Double Labeling ◽

A Cell ◽

Widespread Availability

The widespread availability of automated fluorescence microscope systems has led to an explosion in the acquisition of digital images by biologists. This has created a need for computer applications that automate the analysis of these images and an opportunity to develop new approaches to classical problems. An example is the determination of the subcellular location of a protein from immunofluorescence images (or, more recently, images of GFP fluorescence). Current practice is to compare such images to mental images that a cell biologist has developed over time, and to reach a tentative conclusion about the structure (i.e., organelle) that a protein is found in. Since this determination is subjective, it often must be followed up by double labeling with a marker protein from the suspected structure.As an initial exploration of the feasibility of automating the determination of subcellular location, we developed a system that is able to classify the localization patterns characteristic of five cellular molecules (proteins and DNA) in Chinese Hamster Ovary (CHO) cells. Images were acquired on an epifluorescence microscope after the cells had been fixed, permeabilized, and labeled with appropriate fluorescent reagents (usually antibodies conjugated to fluorescent dyes). The labels used were directed against a Golgi protein, a lysosomal protein, a nuclear protein, a cytoskeletal protein, and DNA.

Download Full-text

Drug targeting to decrease cardiotoxicity – determination of the cytotoxic effect of GnRH-based conjugates containing doxorubicin, daunorubicin and methotrexate on human cardiomyocytes and endothelial cells

Beilstein Journal of Organic Chemistry ◽

10.3762/bjoc.14.136 ◽

2018 ◽

Vol 14 ◽

pp. 1583-1594 ◽

Cited By ~ 4

Author(s):

Livia Polgár ◽

Eszter Lajkó ◽

Pál Soós ◽

Orsolya Láng ◽

Marilena Manea ◽

...

Keyword(s):

Endothelial Cells ◽

Anticancer Drug ◽

Drug Targeting ◽

Cytotoxic Effect ◽

Cell Types ◽

Chemotherapeutic Agents ◽

Limiting Factor ◽

Electrode Arrays ◽

Human Cardiomyocytes

Background: Cardiomyopathy induced by the chemotherapeutic agents doxorubicin and daunorubicin is a major limiting factor for their application in cancer therapy. Chemotactic drug targeting potentially increases the tumor selectivity of drugs and decreases their cardiotoxicity. Increased expression of gonadotropin-releasing hormone (GnRH) receptors on the surface of tumor cells has been reported. Thus, the attachment of the aforementioned chemotherapeutic drugs to GnRH-based peptides may result in compounds with increased therapeutic efficacy. The objective of the present study was to examine the cytotoxic effect of anticancer drug–GnRH-conjugates against two essential cardiovascular cell types, such as cardiomyocytes and endothelial cells. Sixteen different previously developed GnRH-conjugates containing doxorubicin, daunorubicin and methotrexate were investigated in this study. Their cytotoxicity was determined on primary human cardiac myocytes (HCM) and human umbilical vein endothelial cells (HUVEC) using the xCELLigence SP system, which measures impedance changes caused by adhering cells on golden electrode arrays placed at the bottom of the wells. Slopes of impedance–time curves were calculated and for the quantitative determination of cytotoxicity, the difference to the control was analysed. Results: Doxorubicin and daunorubicin exhibited a cytotoxic effect on both cell types, at the highest concentrations tested. Doxorubicin-based conjugates (AN-152, GnRH-III(Dox-O-glut), GnRH-III(Dox-glut-GFLG) and GnRH-III(Dox=Aoa-GFLG) showed the same cytotoxic effect on cardiomyocytes. Among the daunorubicin-based conjugates, [4Lys(Ac)]-GnRH-III(Dau=Aoa), GnRH-III(Dau=Aoa-YRRL), {GnRH-III(Dau=Aoa-YRRL-C)}2 and {[4 N-MeSer]-GnRH-III(Dau-C)}2 had a significant but decreased cytotoxic effect, while the other conjugates – GnRH-III(Dau=Aoa), GnRH-III(Dau=Aoa-K(Dau=Aoa)), [4Lys(Dau=Aoa)]-GnRH-III(Dau=Aoa), GnRH-III(Dau=Aoa-GFLG), {GnRH-III(Dau-C)}2 and [4 N-MeSer]-GnRH-III(Dau=Aoa) – exerted no cytotoxic effect on cardiomyocytes. Mixed conjugates containing methotrexate and daunorubicin – GnRH-III(Mtx-K(Dau=Aoa)) and [4Lys(Mtx)]-GnRH-III(Dau=Aoa) – showed no cytotoxic effect on cardiomyocytes, as well. Conclusion: Based on these results, anticancer drug–GnRH-based conjugates with no cytotoxic effect on cardiomyocytes were identified. In the future, these compounds could provide a more targeted antitumor therapy with no cardiotoxic adverse effects. Moreover, impedimetric cytotoxicity analysis could be a valuable technique to determine the effect of drugs on cardiomyocytes.

Download Full-text