A new fluorescent probe for the equilibrative inhibitor-sensitive nucleoside transporter. 5′-S-(2-aminoethyl)-N6-(4-nitrobenzyl)-5′-thioadenosine (SAENTA)-x2-fluorescein

The N6-(4-nitrobenzyl) derivative of adenosine is a tight-binding inhibitor of the equilibrative inhibitor-sensitive nucleoside transporter of mammalian cells. A fluorescent ligand for this transporter has been synthesized by allowing an adenosine analogue. 5′-S-(2-aminoethyl)-N6-(4-nitrobenzyl)-5′-thioadenosine (SAENTA), to react with fluorescein isothiocyanate. The purified adduct had a SAENTA/fluorescein molar ratio of 0.92:1 calculated from its absorption spectrum. The intensity of fluorescent emission from the SAENTA-chi 2-fluorescein adduct was 30% that of fluorescein isothiocyanate (chi 2 is the number of atoms in the linkage between fluorescein and SAENTA). SAENTA-chi 2-fluorescein inhibited the influx of nucleosides into cultured leukaemic cells with an IC50 (total concentration of inhibitor producing 50% inhibition) of 40 nM. The adduct inhibited the binding of [3H]nitrobenzylthioinosine ([3H]NBMPR) with half-maximal inhibition at 50-100 nM. Mass Law analysis of the competitive-binding data suggested the presence of two classes of sites for [3H]NBMPR binding, only one of which was accessible to SAENTA-chi 2-fluorescein. Flow cytometry was used to analyse equilibrium binding of SAENTA-chi 2-fluorescein to leukaemic cells and a Kd of 6 nM was obtained. SAENTA-chi 2-fluorescein is a high-affinity ligand for the equilibrative inhibitor-sensitive nucleoside transporter which allows rapid assessment of transport capacity by flow cytometry.

Download Full-text

Equilibrative Nucleoside Transporters Mediate the Import of Nicotinamide Riboside and Nicotinic Acid Riboside into Human Cells

International Journal of Molecular Sciences ◽

10.3390/ijms22031391 ◽

2021 ◽

Vol 22 (3) ◽

pp. 1391

Author(s):

Andrey Kropotov ◽

Veronika Kulikova ◽

Kirill Nerinovski ◽

Alexander Yakimov ◽

Maria Svetlova ◽

...

Keyword(s):

Nicotinic Acid ◽

Mammalian Cells ◽

Experimental Models ◽

The Body ◽

Nucleoside Transporters ◽

Hek293 Cells ◽

Nucleoside Transporter ◽

Vitamin B3 ◽

Animal Cells ◽

Nicotinamide Riboside

Nicotinamide riboside (NR), a new form of vitamin B3, is an effective precursor of nicotinamide adenine dinucleotide (NAD+) in human and animal cells. The introduction of NR into the body effectively increases the level of intracellular NAD+ and thereby restores physiological functions that are weakened or lost in experimental models of aging and various pathologies. Despite the active use of NR in applied biomedicine, the mechanism of its transport into mammalian cells is currently not understood. In this study, we used overexpression of proteins in HEK293 cells, and metabolite detection by NMR, to show that extracellular NR can be imported into cells by members of the equilibrative nucleoside transporter (ENT) family ENT1, ENT2, and ENT4. After being imported into cells, NR is readily metabolized resulting in Nam generation. Moreover, the same ENT-dependent mechanism can be used to import the deamidated form of NR, nicotinic acid riboside (NAR). However, NAR uptake into HEK293 cells required the stimulation of its active utilization in the cytosol such as phosphorylation by NR kinase. On the other hand, we did not detect any NR uptake mediated by the concentrative nucleoside transporters (CNT) CNT1, CNT2, or CNT3, while overexpression of CNT3, but not CNT1 or CNT2, moderately stimulated NAR utilization by HEK293 cells.

Download Full-text

Glycoconjugates on the surface of spores of the pathogenic fungus Phytophthora cinnamomi studied using fluorescence and electron microscopy and flow cytometry

Canadian Journal of Microbiology ◽

10.1139/m90-032 ◽

1990 ◽

Vol 36 (3) ◽

pp. 183-192 ◽

Cited By ~ 22

Author(s):

A. R. Hardham ◽

E. Suzaki

Keyword(s):

Flow Cytometry ◽

Plasma Membrane ◽

Cell Surface ◽

Phytophthora Cinnamomi ◽

Pathogenic Fungus ◽

Fluorescein Isothiocyanate ◽

Pathogenic Fungi ◽

Surface Flow ◽

Soybean Agglutinin ◽

Binding Material

Glycoconjugates on the surface of zoospores and cysts of the pathogenic fungus Phytophthora cinnamomi have been studied using fluorescein isothiocyanate labelled lectins for fluorescence microscopy and flow cytometry, and ferritin- and gold-labelled lectins for ultrastructural analysis. Of the five lectins used, only concanavalin A (ConA) binds to the surface of the zoospores, including the flagella and water expulsion vacuole. This suggests that of accessible saccharides, glucosyl or mannosyl residues predominate on the outer surface of the zoospore plasma membrane. Early in encystment, a system of flat disc-like cisternae, which underlie the zoospore plasma membrane, vesiculate. These and other small peripheral vesicles quickly disappear. After the induction of encystment, ConA is no longer localised close to the plasma membrane but binds to material loosely associated with the cell surface. Quantitative measurements by flow cytometry indicate that the ConA-binding material is gradually lost from the cell surface. The cyst wall is weakly labelled, but the site of germ tube emergence stains intensely. During the first 2 min after the induction of encystment, material that binds soybean agglutinin, Helix pommatia agglutinin, and peanut agglutinin appears on the surface of the fungal cells. The distribution of this material, rich in galactosyl or N-acetyl-D-galactosaminosyl residues, is initially patchy, but by 5 min the material evenly coats most of the cell surface. Labelling of zoospores in which intracellular sites are accessible indicates that the soybean agglutinin binding material is stored in vesicles that lie beneath the plasma membrane. Quantitation of soybean agglutinin labelling shows that maximum binding occurs 2–3 min after the induction of encystment. Key words: cell surface, flow cytometry, lectins, pathogenic fungi, Phytophthora cinnamomi.

Download Full-text

Finding the Binding Site of Peloruside A and its Secondary Effects in Saccharomyces Cerevisiae using a Chemical Genetics Approach

10.26686/wgtn.16969795.v1 ◽

2021 ◽

Author(s):

◽

Reem Hanna

Keyword(s):

Saccharomyces Cerevisiae ◽

Cell Cycle ◽

Amino Acids ◽

Flow Cytometry ◽

Binding Site ◽

Mammalian Cells ◽

Site Directed Mutagenesis ◽

Directed Mutagenesis ◽

Peloruside A ◽

Microtubule Function

<p>Peloruside A, a natural product isolated from the marine sponge Mycale hentscheli, is a microtubule-stabilising agent that has a similar mechanism of action to the anticancer drug paclitaxel and is cytotoxic to cultured mammalian cells. Peloruside appears to bind to a distinct site on mammalian tubulin that is different from that of the taxoid-site drugs. Because of the high sequence homology between yeast and mammalian tubulin, Saccharomyces cerevisiae (S. cerevisiae) was used as a model organism to characterise the peloruside-binding site with the aim of advancing our understanding about this site on mammalian tubulin. Wild type S. cerevisiae (BY4741) was sensitive to peloruside at uM concentrations; however, a strain that lacks the mad2 (Mitotic Arrest Deficient 2) gene showed increased sensitivity to the drug at much lower uM concentrations. This gene is a component of the spindle-assembly checkpoint complex that delays the onset of anaphase in cells with defects in mitotic spindle assembly. The main aims of this project were to define the binding site of peloruside A using yeast tubulin to see if microtubule function and/or morphology is altered in yeast by peloruside, and to identify any secondary drug targets "friends of the target" through chemical genetic interactions profiling (Homozygous deletion profiling microarray). Site-directed mutagenesis was used to mutate two conserved amino acids (A296T; R306H) known to confer resistance to peloruside in mammalian cells. Based on a published computer model of the peloruside binding site on mammalian tubulin, we also mutated three other amino acids, two that were predicted to affect peloruside binding (Q291M and N337L), and one that was predicted to affect laulimalide binding but have little affect on peloruside binding (V333W). We also included a negative control that was predicted to have no effect on peloruside binding (R282Q) and would affect epothilone binding. We found that of the six point mutations, only Q291M failed to confer resistance in yeast and instead it increased the inhibition to the drug. Using a bud index assay, confocal microscopy, and flow cytometry, 40-50 uM peloruside was shown to block cells in G2/M of the cell cycle, confirming a direct action of the drug on microtubule function. Homozygous profiling (HOP) microarray analysis of a deletion mutant set of yeast genes was also carried out to identify gene products that interact with peloruside in order to link the drug to specific networks or biochemical pathways in the cells. From site-directed mutagenesis, we concluded that peloruside binds to yeast B-tubulin in the region predicted by the published model of the binding site, and therefore mapping the site on yeast tubulin could provide useful information about the mammalian binding site for peloruside. The bud index, flow cytometry, and confocal microscopy experiments provided further evidence that peloruside interacts with yeast tubulin. From HOP we found that peloruside has roles in the cell cycle, as expected, and has effects on protein transport, secretion, cell wall synthesis, and steroid biosynthesis pathways.</p>

Download Full-text

Glyoxal fixation facilitates transcriptome analysis after antigen staining and cell sorting by flow cytometry

PLoS ONE ◽

10.1371/journal.pone.0240769 ◽

2021 ◽

Vol 16 (1) ◽

pp. e0240769

Author(s):

Prasanna Channathodiyil ◽

Jonathan Houseley

Keyword(s):

Flow Cytometry ◽

Transcriptome Analysis ◽

Mammalian Cells ◽

Single Cells ◽

Rna Extraction ◽

Cyclin B1 ◽

Immunofluorescent Staining ◽

Rna Seq ◽

Simple Method ◽

Staining Methods

A simple method for extraction of high quality RNA from cells that have been fixed, stained and sorted by flow cytometry would allow routine transcriptome analysis of highly purified cell populations and single cells. However, formaldehyde fixation impairs RNA extraction and inhibits RNA amplification. Here we show that good quality RNA can be readily extracted from stained and sorted mammalian cells if formaldehyde is replaced by glyoxal—a well-characterised fixative that is widely compatible with immunofluorescent staining methods. Although both formaldehyde and glyoxal efficiently form protein-protein crosslinks, glyoxal does not crosslink RNA to proteins nor form stable RNA adducts, ensuring that RNA remains accessible and amenable to enzymatic manipulation after glyoxal fixation. We find that RNA integrity is maintained through glyoxal fixation, permeabilisation with methanol or saponin, indirect immunofluorescent staining and flow sorting. RNA can then be extracted by standard methods and processed into RNA-seq libraries using commercial kits; mRNA abundances measured by poly(A)+ RNA-seq correlate well between freshly harvested cells and fixed, stained and sorted cells. We validate the applicability of this approach to flow cytometry by staining MCF-7 cells for the intracellular G2/M-specific antigen cyclin B1 (CCNB1), and show strong enrichment for G2/M-phase cells based on transcriptomic data. Switching to glyoxal fixation with RNA-compatible staining methods requires only minor adjustments of most existing staining and sorting protocols, and should facilitate routine transcriptomic analysis of sorted cells.

Download Full-text

Identification, characterization and localization of chagasin, a tight-binding cysteine protease inhibitor inTrypanosoma cruzi

Journal of Cell Science ◽

10.1242/jcs.114.21.3933 ◽

2001 ◽

Vol 114 (21) ◽

pp. 3933-3942 ◽

Cited By ~ 4

Author(s):

Ana C. S. Monteiro ◽

Magnus Abrahamson ◽

Ana P. C. A. Lima ◽

Marcos A. Vannier-Santos ◽

Julio Scharfstein

Keyword(s):

Cell Surface ◽

Protease Inhibitor ◽

Cysteine Protease ◽

Mammalian Cells ◽

Tight Binding ◽

Cysteine Proteases ◽

Host Cells ◽

Cysteine Protease Inhibitor ◽

Reversible Inhibitor ◽

Mammalian Host

Lysosomal cysteine proteases from mammalian cells and plants are regulated by endogenous tight-binding inhibitors from the cystatin superfamily. The presence of cystatin-like inhibitors in lower eukaryotes such as protozoan parasites has not yet been demonstrated, although these cells express large quantities of cysteine proteases and may also count on endogenous inhibitors to regulate cellular proteolysis. Trypanosoma cruzi, the causative agent of Chagas’ heart disease, is a relevant model to explore this possibility because these intracellular parasites rely on their major lysosomal cysteine protease (cruzipain) to invade and multiply in mammalian host cells. Here we report the isolation, biochemical characterization, developmental stage distribution and subcellular localization of chagasin, an endogenous cysteine protease inhibitor in T. cruzi. We used high temperature induced denaturation to isolate a heat-stable cruzipain-binding protein (apparent molecular mass, 12 kDa) from epimastigote lysates. This protein was subsequently characterized as a tight-binding and reversible inhibitor of papain-like cysteine proteases. Immunoblotting indicated that the expression of chagasin is developmentally regulated and inversely correlated with that of cruzipain. Gold-labeled antibodies localized chagasin to the flagellar pocket and cytoplasmic vesicles of trypomastigotes and to the cell surface of amastigotes. Binding assays performed by probing living parasites with fluorescein (FITC)-cruzipain or FITC-chagasin revealed the presence of both inhibitor and protease at the cell surface of amastigotes. The intersection of chagasin and cruzipain trafficking pathways may represent a checkpoint for downstream regulation of proteolysis in trypanosomatid protozoa.

Download Full-text

In Vitro Testing of a New Substance with Anti-Tumor Activity on Mammalian Cells Using Flow Cytometry

Animal Cell Technology: From Target to Market ◽

10.1007/978-94-010-0369-8_1 ◽

2001 ◽

pp. 3-5

Author(s):

Arne Burzlaff ◽

Cornelia Kasper ◽

Mathias Christmann ◽

Ulhas Bhatt ◽

Markus Kalesse ◽

...

Keyword(s):

Flow Cytometry ◽

Mammalian Cells ◽

In Vitro Testing ◽

Anti Tumor Activity

Download Full-text

Effect of single oral dose of azithromycin, clarithromycin, and roxithromycin on polymorphonuclear leukocyte function assessed ex vivo by flow cytometry.

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.40.9.2039 ◽

1996 ◽

Vol 40 (9) ◽

pp. 2039-2042 ◽

Cited By ~ 12

Author(s):

C Wenisch ◽

B Parschalk ◽

K Zedtwitz-Liebenstein ◽

A Weihs ◽

I el Menyawi ◽

...

Keyword(s):

Flow Cytometry ◽

Oral Dose ◽

Single Oral Dose ◽

Ex Vivo ◽

Reactive Oxygen ◽

Fluorescein Isothiocyanate ◽

Neutrophil Granulocytes ◽

Oxygen Production ◽

Phagocytic Cells ◽

Phagocytic Capacity

Azithromycin was given as a single oral dose (20 mg/kg of body weight) to 12 volunteers in a crossover study with roxithromycin (8 to 12 mg/kg) and clarithromycin (8 to 12 mg/kg). Flow cytometry was used to study the phagocytic functions and the release of reactive oxygen products following phagocytosis by neutrophil granulocytes prior to administration of the three drugs, 16 h after azithromycin administration, and 3 h after clarithromycin and roxithromycin administration. Phagocytic capacity was assessed by measuring the uptake of fluorescein isothiocyanate-labeled bacteria. Reactive oxygen generation after phagocytosis of unlabeled bacteria was estimated by the amount of dihydrorhodamine 123 converted to rhodamine 123 intracellularly. Azithromycin resulted in decreased capacities of the cells to phagocytize Escherichia coli (median [range], 62% [27 to 91%] of the control values; P < 0.01) and generate reactive oxygen products (75% [34 to 26%] of the control values; P < 0.01). Clarithromycin resulted in reduced phagocytosis (82% [75 to 98%] of control values; P < 0.01) but did not alter reactive oxygen production (84% [63 to 113%] of the control values; P > 0.05). Roxithromycin treatment did not affect granulocyte phagocytosis (92% [62 to 118%] of the control values; P > 0.05) or reactive oxygen production (94% [66 to 128%] of the control value; P > 0.05). No relation between intra- and/or extracellular concentrations of azithromycin and/or roxithromycin and the polymorphonuclear phagocyte function and/or reactive oxygen production existed (P > 0.05 for all comparisons). These results demonstrate that the accumulation of macrolides in neutrophils can suppress the response of phagocytic cells to bacterial pathogens after a therapeutic dose.

Download Full-text

Fabrication of Superhydrophobic Films on Aluminum Foils with Controllable Morphologies

Advanced Materials Research ◽

10.4028/www.scientific.net/amr.641-642.414 ◽

2013 ◽

Vol 641-642 ◽

pp. 414-417 ◽

Cited By ~ 2

Author(s):

Yu Zhen Lv ◽

Le Feng Wang ◽

Kai Bo Ma ◽

You Zhou ◽

Cheng Rong Li

Keyword(s):

Contact Angle ◽

Water Contact Angle ◽

Solution Method ◽

Total Concentration ◽

Zinc Nitrate ◽

Zinc Nitrate Hexahydrate ◽

Simple Solution ◽

Molar Ratio ◽

Nitrate Hexahydrate ◽

Water Contact

In this study, porous architectures with controllable morphologies on Aluminum foils were fabricated by a simple solution method using zinc nitrate hexahydrate and hexamethylenetetramine as reactants. Unlike works reported previously, we show that the porous architectures on the surface of Aluminum foils can be adjusted by controlling the molar ratio and total concentration of reactants, which is proved to be of importance in tailoring the wettability of the surface. It is found that by adjusting the space of pores and the thickness of nanoslices, the water contact angle of the obtained surface can change from hydrophobic to superhydrophobic.

Download Full-text