A novel electrochemical conducting polymer sensor for the rapid, selective and sensitive detection of biothiols

2021 ◽  
Author(s):  
Bicheng Zhu ◽  
Devon Bryant ◽  
Alireza Akbarinejad ◽  
Jadranka Travas-Sejdic ◽  
Lisa I Pilkington
Keyword(s):  
Conducting Polymer ◽  
Sensitive Detection ◽  
Bodily Fluids ◽  
Wide Range ◽  
In Cells

Biological thiols (biothiols) in cells and bodily fluids are intrinsically linked to the functioning of important enzymes, deficiency in which can lead to a wide range of physiological and pathological...

Two-Color Probe to Monitor a Wide Range of pH Values in Cells

2013 ◽  
Vol 52 (24) ◽  
pp. 6206-6209 ◽  
Author(s):  
Min Hee Lee ◽  
Ji Hye Han ◽  
Jae Hong Lee ◽  
Nayoung Park ◽  
Rajesh Kumar ◽  
...  
Keyword(s):  
Ph Values ◽  
Wide Range ◽  
In Cells

Two-Color Probe to Monitor a Wide Range of pH Values in Cells

2013 ◽  
Vol 125 (24) ◽  
pp. 6326-6329 ◽  
Author(s):  
Min Hee Lee ◽  
Ji Hye Han ◽  
Jae Hong Lee ◽  
Nayoung Park ◽  
Rajesh Kumar ◽  
...  
Keyword(s):  
Ph Values ◽  
Wide Range ◽  
In Cells

scholarly journals A validated collection of mouse monoclonal antibodies to human glycosyltransferases functioning in mucin-type O-glycosylation

Glycobiology ◽  
2019 ◽  
Vol 29 (9) ◽  
pp. 645-656 ◽  
Author(s):  
Catharina Steentoft ◽  
Zhang Yang ◽  
Shengjun Wang ◽  
Tongzhong Ju ◽  
Malene B Vester-Christensen ◽  
...  
Keyword(s):  
Monoclonal Antibodies ◽  
Secretory Pathway ◽  
Expression Patterns ◽  
In Situ Monitoring ◽  
Kinetic Properties ◽  
Detailed Knowledge ◽  
Cellular Expression ◽  
Wide Range ◽  
Catalytically Active ◽  
In Cells

Abstract Complex carbohydrates serve a wide range of biological functions in cells and tissues, and their biosynthesis involves more than 200 distinct glycosyltransferases (GTfs) in human cells. The kinetic properties, cellular expression patterns and subcellular topology of the GTfs direct the glycosylation capacity of a cell. Most GTfs are ER or Golgi resident enzymes, and their specific subcellular localization is believed to be distributed in the secretory pathway according to their sequential role in the glycosylation process, although detailed knowledge for individual enzymes is still highly fragmented. Progress in quantitative transcriptome and proteome analyses has greatly advanced our understanding of the cellular expression of this class of enzymes, but availability of appropriate antibodies for in situ monitoring of expression and subcellular topology have generally been limited. We have previously used catalytically active GTfs produced as recombinant truncated secreted proteins in insect cells for generation of mouse monoclonal antibodies (mAbs) to human enzymes primarily involved in mucin-type O-glycosylation. These mAbs can be used to probe subcellular topology of active GTfs in cells and tissues as well as their presence in body fluids. Here, we present several new mAbs to human GTfs and provide a summary of our entire collection of mAbs, available to the community. Moreover, we present validation of specificity for many of our mAbs using human cell lines with CRISPR/Cas9 or zinc finger nuclease (ZFN) knockout and knockin of relevant GTfs.

scholarly journals A fluorescent methylation-switchable probe for highly sensitive analysis of FTO N6-methyladenosine demethylase activity in cells

2018 ◽  
Vol 9 (36) ◽  
pp. 7174-7185 ◽  
Author(s):  
Adeline Cheong ◽  
Joanne J. A. Low ◽  
Andrea Lim ◽  
Paul M. Yen ◽  
Esther C. Y. Woon
Keyword(s):  
Methylation Status ◽  
Sensitive Detection ◽  
Sensitive Analysis ◽  
Highly Sensitive ◽  
Demethylase Activity ◽  
Highly Sensitive Detection ◽  
In Cells

A dynamic biosensor which switches conformation according to its methylation status enables highly sensitive detection of m6A-demethylase activity. This strategy may be adapted to a broad range of RNA-modifying enzymes.

STAT3 Activates Anti-Apoptotic Genes But When In Access Induces Apoptosis Of CLL Cells

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 5294-5294 ◽  
Author(s):  
Rozovski Uri ◽  
David Harris ◽  
Ping Li ◽  
Zhiming Liu ◽  
Ji Yuan ◽  
...  
Keyword(s):  
Binding Affinity ◽  
Caspase 3 ◽  
Conflicts Of Interest ◽  
Lymphocytic Leukemia ◽  
Luciferase Assay ◽  
Spontaneous Apoptosis ◽  
Mobility Shift ◽  
Apoptotic Pathway ◽  
Wide Range ◽  
In Cells

Abstract In chronic lymphocytic leukemia (CLL) cells, signal transducer and activator of transcription 3 (STAT3) is constitutively phosphorylated on serine 727 residues, and phosphoserine STAT3 induces the transcription and proliferation of anti-apoptosis genes including c-Myc, BCL2, Bcl-XL, and Mcl-1 (Hazan-Halevy, 2010). Therefore, we hypothesized that the CLL cells of patients with high peripheral blood absolute lymphocyte counts (ALCs) have a high level of STAT3 protein expression and a low rate of spontaneous apoptosis. Using Western blotting and flow cytometry, we quantitated STAT3 levels and apoptosis rates in CLL cells from 64 patients with high (N=32) and low (N=32) ALCs. As expected, the levels of STAT3 expression were significantly higher in cells from patients with high ALCs (145,000 ± 49,738/µL) than in cells from patients with low ALCs (12,800 ± 4,654/µL). However, contrary to our hypothesis, Annexin V/PI staining revealed that the levels of procaspase-3, its activated (cleaved) form caspase-3, and cleaved PARP as well as the apoptosis rates of cells from patients with high ALCs were significantly higher than those in cells from patients with low ALCs. These findings suggest that cells from patients with high ALCs are prone to spontaneous apoptosis. An RNA microarray analysis revealed that the levels of apoptotic pathway genes were upregulated in cells from patients with high ALC (P = 0.002), and Reverse-transcriptaction PCR of Caspase-3, Calpain 9, MAPK 8, KRAS, PLCγ-2, and PKC validated the array data. Intrigued by these findings, we sought to determine whether high levels of intracellular STAT3 induce apoptosis. Indeed, overexpression of STAT3 in interleukin-6–stimulated MM1 cells upregulated caspase-3 and caspase-3 protein levels and induced apoptosis. Similarly, in CLL cells levels of caspase3 and procaspase3 remained stable across a wide range of STAT3 levels, but when STAT3 reached a threshold, level of caspase3 and procaspase3 markedly increased. Because sequence analysis revealed that the caspase-3 promoter harbors γ-interferon activation sequence-like elements, we cloned the caspase-3 promoter in MM1 cells and, using a luciferase assay, identified regions with putative STAT3 binding sites. Chromatin immunoprecipitation (ChIP) and an electrophoretic mobility shift essay (EMSA) confirmed that STAT3 binds to the promoter of caspase-3. However, caspase-3 was activated only in cells with high STAT3 expression levels, suggesting that STAT3 binds to caspase-3 with low affinity. To assess STAT3’s binding affinity to the caspase-3 promoter, we prepared serial dilutions of CLL cell DNA and, using ChIP and EMSA, found that STAT3’s binding affinities to p21 and c-Myc were 8 and 4 times as high, respectively, as its binding affinity to caspase-3, suggesting that high levels of STAT3 protein are required to activate caspase-3. Taken together, these findings suggest that activated STAT3 has a previously unknown pro-apoptotic function. At high intracellular levels, rather than providing CLL cells with survival advantage, STAT3 induces apoptosis by activating caspase-3. Thus, because CLL cell proliferation is coupled with spontaneous CLL cell apoptosis, the number of circulating CLL cells based on the cells’ proliferation rate is often lower than expected. Disclosures: No relevant conflicts of interest to declare.

scholarly journals Enhancement of homology-directed repair with chromatin donor templates in cells

eLife ◽  
2020 ◽  
Vol 9 ◽  
Author(s):  
Grisel Cruz-Becerra ◽  
James T Kadonaga
Keyword(s):  
Genome Editing ◽  
Dna Fragments ◽  
Natural Form ◽  
Naked Dna ◽  
Homology Directed Repair ◽  
Coding Regions ◽  
Wide Range ◽  
Low Efficiency ◽  
Donor Template ◽  
In Cells

A key challenge in precise genome editing is the low efficiency of homology-directed repair (HDR). Here we describe a strategy for increasing the efficiency of HDR in cells by using a chromatin donor template instead of a naked DNA donor template. The use of chromatin, which is the natural form of DNA in the nucleus, increases the frequency of HDR-edited clones as well as homozygous editing. In addition, transfection of chromatin results in negligible cytotoxicity. These findings suggest that a chromatin donor template should be useful for a wide range of HDR applications such as the precise insertion or replacement of DNA fragments that contain the coding regions of genes.

scholarly journals Synthesis of new conducting materials on the basis of polymer hydrogels

2008 ◽  
Vol 2 (2) ◽  
pp. 99-104
Author(s):  
Oleh Suberlyak ◽  
◽  
Oleksandr Hrytsenko ◽  
Khrystyna Hishchak ◽  
◽  
...  
Keyword(s):  
Magnetic Field ◽  
Moisture Content ◽  
Ambient Temperature ◽  
Conducting Polymer ◽  
Synthesis Conditions ◽  
Polymer Hydrogels ◽  
Conducting Materials ◽  
Powder Filler ◽  
Wide Range ◽  
Field Action

The new conducting polymer hydrogels on the basis of co-polymers of hydroxyethylenemethacrylate and polyvinylpyrrolidone with different nature non-organic fillers have been developed. The dependence of obtained materials electric characteristics on synthesis conditions, quantity and nature of powder filler, moisture content, ambient temperature and magnetic field action have been determined. The possibility of obtaining materials with anisotropic and unidirectional conductivity as well as the wide range of conductivity, which changes with moisture and ambient temperature, has been considered in this work

Measurements of Ca 2+ fluxes in intact plant cells

1992 ◽  
Vol 338 (1283) ◽  
pp. 73-82 ◽  
Keyword(s):  
Signal Transduction ◽  
Direct Method ◽  
Plant Cells ◽  
Giant Cells ◽  
Chara Corallina ◽  
Resting Cells ◽  
Cytosolic Ph ◽  
Wide Range ◽  
Cell Turgor ◽  
In Cells

Owing to the central role of Ca 2+ in signal transduction processes, it is important to measure membrane fluxes of Ca 2+ in cells which are as undisturbed as possible, particularly when studying the control of these fluxes. To this end, techniques have been developed to measure Ca 2+ fluxes in intact, turgid plant cells. The measurements are principally of influx across the plasma membrane where Ca 2+ transport is likely to occur through cation-selective channels. The most direct method measures tracer fluxes of Ca 2+ , but special procedures are required to distinguish between influx and extracellular binding of Ca 2+ . Unfortunately, such techniques are currently only applicable to giant cells where surgical separation of the intracellular contents from the cell wall is possible. The influx of Ca 2+ into normal, resting cells of the green alga Chara corallina is usually about 0.3 nmol m -2 s -1 (at an external Ca 2+ concentration of 0.5 mol m -3 ). This flux is up to 5 times higher in actively growing cells, 20 times higher in cells depolarized by 20 mol m -3 K + and 1000 times higher during an action potential. Reducing cell turgor by a wide range of solutes increases Ca 2+ influx, especially near plasmolysis. Ca 2+ influx is sensitive to alterations in both external and cytosolic pH, but is inhibited by complete darkness and by low concentrations of La 3+ . Various organic Ca 2+ channel antagonists had mixed effects on Ca 2+ influx into Chara . The work described in this paper should enable further study of the control of Ca 2+ fluxes into intact, turgid plant cells, and their role in signal transduction and the control of cellular activities.

scholarly journals Effect of weak bases on the intralysosomal pH in mouse peritoneal macrophages.

1981 ◽  
Vol 90 (3) ◽  
pp. 665-669 ◽  
Author(s):  
B Poole ◽  
S Ohkuma
Keyword(s):  
Fluorescence Probe ◽  
Spectral Characteristics ◽  
Active Form ◽  
Peritoneal Macrophages ◽  
Weak Bases ◽  
Wide Range ◽  
A Minor ◽  
Energy Dependent ◽  
Mouse Peritoneal Macrophages ◽  
In Cells

The spectral characteristics of dextran, labeled with fluorescein, depend upon pH. We have loaded the lysosomes of mouse peritoneal macrophages with this fluorescence probe and used it to measure the intralysosomal pH under various conditions. The pH of the medium has no effect on the intralysosomal pH. Weakly basic substances in the medium cause a concentration-dependent increase in the intralysosomal pH. However, the concentration of base necessary to produce a significant change in the intralysosomal pH varies over a wide range for different bases. The active form of the base is the neutral, unprotonated form. Although most of these weak bases cause an increase in the volume of the lysosomes, increase in lysosomal volume itself causes only a minor perturbation of the intralysosomal pH. This was demonstrated in cells whose lysosomes were loaded with sucrose, and in cells vacuolated as a demonstrated in cells whose lysosomes were loaded with sucrose, and in cells vacuolated as a consequence of exposure to concanavalin A. The results of these studies are interpreted in terms of energy-dependent lysosomal acidification and leakage of protons out of the lysosomes in the form of protonated weak bases.

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