scholarly journals Development of selective high-affinity fluorescent sensors for imaging neurotransmitters and application in cells and tissue

2019 ◽  
Author(s):  
◽  
Le Zhang
Keyword(s):  
Fluorescent Sensor ◽  
Photoreceptor Cells ◽  
Fluorescent Sensors ◽  
Tirf Microscopy ◽  
Glutamatergic Neurons ◽  
High Affinity ◽  
Iminium Ion ◽  
The Difference ◽  
Fluorescence Titrations ◽  
In Cells

Herein, we develop a series of selective fluorescent sensors based on a quinolone core designed to image neurotransmitters catecholamines, serotonin and glutamate. Included are the main design strategy, synthesis, spectroscopic data (UV/Vis and fluorescence) and their applications in cells and tissue. NeuroSensor 510 (NS510) was designed and synthesized as a selective sensor for imaging norepinephrine. Formation of both an iminium ion and a boronate ester gave high affinity upon binding NS510 to norepinephrine. The sensor was able to label norepinephrine vesicles in chromaffin cells to give punctate staining. Moreover, the sensor was able to monitor norepinephrine exocytosis via correlation of total internal reflective fluorescence (TIRF) microscopy and amperometry. A NIR fluorescent sensor NS659 was developed based on the structure of NS510. Several different synthetic routes were attempted before the sensor was successfully prepared. Upon binding to serotonin, the sensor gave a relatively high affinity but with quenched fluorescence. Initial tests showed that the sensor could be used to label vesicles. Finally, a molecular sensor NS560 was developed to image glutamate. Fluorescence titrations showed that the sensor bound to glutamate to give a very large turn-on response. More importantly, the sensor should be able to differentiate glutamate from GABA due to the difference both in the emission wavelength and the fluorescence response. We tested this sensor in photoreceptor cells and observed that vesicles were labeled by this sensor. Destaining of the vesicles was observed via TIRF microscopy. Also, the sensor can label glutamate in cultured glutamatergic neurons, astrocytes and cortex brain.

Detection of boronic acid derivatives in cells using a fluorescent sensor

2015 ◽  
Vol 13 (25) ◽  
pp. 6927-6930 ◽  
Author(s):  
Yoshihide Hattori ◽  
Miki Ishimura ◽  
Youichirou Ohta ◽  
Hiroshi Takenaka ◽  
Tsubasa Watanabe ◽  
...  
Keyword(s):  
Boronic Acid ◽  
Detection Method ◽  
Fluorescent Sensor ◽  
Fluorescent Sensors ◽  
Chelating Ligands ◽  
Boronic Acid Derivatives ◽  
In Cells

To develop a detection method for boronic acid derivatives, boron-chelating ligands were synthesized as fluorescent sensors for boronic acid derivatives.

Mitochondria-targeting fluorescent sensor for on-off-on response to Cu2+ and ATP in cells and zebrafish

The Analyst ◽  
2021 ◽  
Author(s):  
Wan Sun ◽  
Guofeng Liu ◽  
Mingqiong Tong ◽  
Haozhan Wang ◽  
Shuhan Liu
Keyword(s):  
Adenosine Triphosphate ◽  
Fluorescent Sensor ◽  
Biological Processes ◽  
Fluorescent Molecule ◽  
Cupric Ion ◽  
Mitochondria Targeting ◽  
In Cells

Cupric ion (Cu2+) and adenosine triphosphate (ATP) are functionally important in mitochondria and play essential roles in many important biological processes. In this work, a mitochondria-targeting fluorescent molecule Mito-A was...

scholarly journals Mediation of interleukin-11-dependent biological responses by a soluble form of the interleukin-11 receptor

1996 ◽  
Vol 318 (2) ◽  
pp. 489-495 ◽  
Author(s):  
Julia KAROW ◽  
Keith R. HUDSON ◽  
Mark A. HALL ◽  
Ann B. VERNALLIS ◽  
Jacky A. TAYLOR ◽  
...  
Keyword(s):  
Biological Effects ◽  
Biological Response ◽  
Cytoplasmic Domain ◽  
Soluble Form ◽  
High Affinity ◽  
Biological Responses ◽  
Interleukin 11 ◽  
Biological Actions ◽  
In Cells

Interleukin-11 (IL-11) is a polyfunctional cytokine whose biological actions require a specific IL-11 receptor (IL-11R) and the transmembrane transducer gp130. Here we report the production of a soluble form of the murine IL-11R and demonstrate that it interacts with IL-11 ligand with high affinity. The affinity of IL-11 alone for gp130 is below the level of detection, but a complex of IL-11 and soluble IL-11R interacts with gp130 with high affinity. The addition of soluble IL-11R potentiates the effects of exogenous IL-11 in cells that are normally responsive to IL-11. A biological response to IL-11 can be reconstituted in BAF cells transfected with gp130 by addition of IL-11 and soluble IL-11R. These findings show that the cytoplasmic domain of the IL-11R is not required for the biological effects of IL-11 and that a complex of IL-11 and IL-11R mediates signalling by association with gp130.

Sodium current function in adult and aged canine atrial cells

2006 ◽  
Vol 291 (2) ◽  
pp. H756-H761 ◽  
Author(s):  
Shigeo Baba ◽  
Wen Dun ◽  
Masanori Hirose ◽  
Penelope A. Boyden
Keyword(s):  
Normal Sinus Rhythm ◽  
Sodium Current ◽  
Cell Voltage ◽  
Current Function ◽  
Structural Remodeling ◽  
Channel Protein ◽  
Normal Sinus ◽  
Atrial Cells ◽  
The Difference ◽  
In Cells

The incidence of atrial fibrillation increases with age, but it is unknown whether there are changes in the intrinsic function of Na+ currents in cells of the aged atria. Thus, we studied right (RA) and left (LA) atrial cells from two groups of dogs, adult and aged (>8 yr), to determine the change in Na+ currents with age. In this study all dogs were in normal sinus rhythm. Whole cell voltage clamp techniques were used to compare the Na+ currents in the two cell groups. Immunocytochemical studies were completed for the Na+ channel protein Nav1.5 to determine whether there was structural remodeling of this protein with age. In cells from aged animals, we found that Na+ currents are similar to those we measured in adult atria. However, Na+ current ( INa) density of the aged atria differed depending on the atrial chamber with LA cell currents being larger than RA cell currents. Thus with age, the difference in INa density between atrial chambers remains. INa kinetic differences between aged and adult cells included a significant acceleration into the inactivated state and an enhanced use-dependent decrease in peak current in aged RA cells. Finally, there is no structural remodeling of the cardiac Na+ channel protein Nav1.5 in the aged atrial cell. In conclusion, with age there is no change in INa density, but there are subtle kinetic differences contributing to slight enhancement of use dependence. There is no structural remodeling of the fast Na+ current protein with age.

scholarly journals The Effect of Aloe Vera Extract to Extensive Lesion and Expression of Transforming Growth Factor (TGF-β) on Alkaline Chemical Trauma Cornea Model

2018 ◽  
Vol 1 (2) ◽  
pp. 1-17
Author(s):  
Fahcreza ◽  
Elsa Iskandar ◽  
Rachmat Hidayat ◽  
Petty Purwanita ◽  
Anang Tribowo ◽  
...  
Keyword(s):  
Wound Healing ◽  
Growth Factor ◽  
Wistar Rats ◽  
Aloe Vera ◽  
Control Group ◽  
Treatment Groups ◽  
White Rats ◽  
Significant Difference ◽  
The Difference ◽  
In Cells

Abstract Background: Chemical trauma to the cornea is an emergency condition of the eye that requires early diagnosis and good treatment. Alkaline have ability to saponify fatty acids in cells and cell membranes which can make penetration into the stroma and destroy proteoglycans and collagen in cells. Aloe vera (AV) contains several active substances that are reported to have anti-inflammatory, immunomodulatory, and wound healing effects. AV has been reported to accelerate the healing process of corneal epithelial defects by increasing fibroblast proliferation, collagen production and growth factor production. This study aims to determine the difference between the effect of aloe vera extract with a concentration of 10%, 20%, 40% and BSS on the healing of extensive corneal lesions in white wistar rats alkaline trauma models. Method: This study was an experimental study with a pre and posttest only with control group design in vivo approach to 30 Wistar white rats which were divided into 5 treatment groups for 3 days. Comparative analysis of effectiveness using the ANNOVA test or the Kruskal Wallis test and continued by the post hoc test. Results: Based on the one way ANOVA test there was a statistically significant difference in effectiveness between the five treatment groups on the percentage of corneal wound healing area and TGF-β expression with an assessment of p = 0,000 each. The administration of alloevera (AV) concentration of 20% had a significant difference in percentage of healing of corneal lesions and TGF-β expression compared with other treatment groups with p = 0,000 each. Large differences in the area of corneal lesions in the 40% AV group were -0.45 in the BBS group, 0.146 in the 10% AV group, 0.493 in the 20% AV group. The difference in the AV group 10% was 0.30 in the BBS group, -064 in the AV group 20%, and -0.14 in the AV group 40%. However, TGFβ expression in the normal control group that did not receive treatment was 54.94 (53.21-56-12). TGFβ levels in the BSS group were 10.44, the 10% aloe vera group was 25.43, 47.99 for the 20% aloe vera group and 37.95 for the 40% aloe vera group. Conclusion: There is a difference between the effect of aloe vera extract with concentrations of 10%, 20%, 40% and BSS on the extensive healing of corneal lesions in white wistar rats with alkaline chemical trauma models.

Development of highly selective fluorescent sensors for detection of glycolipids in vesicles and visualization of glutamate for neuronal imaging

2020 ◽  
Author(s):  
◽  
Ming Xu
Keyword(s):  
Fluorescence Enhancement ◽  
Chemical Sensor ◽  
Fluorescent Sensor ◽  
Hydrophobic Effect ◽  
Fluorescent Sensors ◽  
Nucleophilic Aromatic Substitution ◽  
Sensor System ◽  
Aromatic Substitution ◽  
Glutamate Binding ◽  
A Cell

[ACCESS RESTRICTED TO THE UNIVERSITY OF MISSOURI-COLUMBIA AT REQUEST OF AUTHOR.] Fluorescent sensors are very useful tools for exploring chemical biology and advanced medical research. Herein, we propose four different fluorescent sensor systems for the recognition of some important biological molecules. The first sensor system is a multi-component fluorescent sensor complex for the sensing of glycolipids. The glycolipid sensor system is a novel design that takes advantage of supramolecular self-assembly. Results show that it can bind with both the sugar headgroup and hydrocarbon tail of glycolipids, and turn on the fluorescence of the sensor system. The second sensor is a cell-impermeable fluorescent sensor system for the recognition and extraction of glycolipids from vesicles. To avoid the fluorescence enhancement caused by the hydrophobic effect from cell membrane, we designed a series of cell-impermeable sensor complexes. In addition, these complexes were fully explored by vesicle studies. Another fluorescent sensor is NS600 which was developed for detecting and imaging glutamate in neurons. This sensor system that utilizes a nucleophilic aromatic substitution for glutamate binding, and produces a 270-fold fluorescence enchantment upon glutamate binding. Also, it overcomes drawbacks of previous glutamate sensors including low signal response and poor sensitivity. It enables a clear and accurate visualization of glutamate in cultural neurons. The last sensor is NS570, a cell-impermeable glutamate sensor which could be loaded into synaptic vesicles by vesicle cycling. This sensor is a reversible chemical sensor that gives a 2600-fold fluorescence enhancement upon the titration with glutamate and can be used to monitor the release of neuronal glutamate in real time.

scholarly journals Microtubule minus-end stability is dictated by the tubulin off-rate

2019 ◽  
Vol 218 (9) ◽  
pp. 2841-2853 ◽  
Author(s):  
Claire Strothman ◽  
Veronica Farmer ◽  
Göker Arpağ ◽  
Nicole Rodgers ◽  
Marija Podolski ◽  
...  
Keyword(s):  
Dynamic Instability ◽  
In Vitro Reconstitution ◽  
Primary Determinant ◽  
Mean Size ◽  
The Mean ◽  
The Difference ◽  
Insight Into ◽  
Dynamic Organization ◽  
In Cells

Dynamic organization of microtubule minus ends is vital for the formation and maintenance of acentrosomal microtubule arrays. In vitro, both microtubule ends switch between phases of assembly and disassembly, a behavior called dynamic instability. Although minus ends grow slower, their lifetimes are similar to those of plus ends. The mechanisms underlying these distinct dynamics remain unknown. Here, we use an in vitro reconstitution approach to investigate minus-end dynamics. We find that minus-end lifetimes are not defined by the mean size of the protective GTP-tubulin cap. Rather, we conclude that the distinct tubulin off-rate is the primary determinant of the difference between plus- and minus-end dynamics. Further, our results show that the minus-end–directed kinesin-14 HSET/KIFC1 suppresses tubulin off-rate to specifically suppress minus-end catastrophe. HSET maintains its protective minus-end activity even when challenged by a known microtubule depolymerase, kinesin-13 MCAK. Our results provide novel insight into the mechanisms of minus-end dynamics, essential for our understanding of microtubule minus-end regulation in cells.

High affinity L-aspartate transport in chick small intestine

1987 ◽  
Vol 252 (1) ◽  
pp. C105-C114 ◽  
Author(s):  
T. G. Wingrove ◽  
G. A. Kimmich
Keyword(s):  
Small Intestine ◽  
Membrane Potential ◽  
Epithelial Cells ◽  
Intracellular Ph ◽  
Transport Function ◽  
Flux Enhancement ◽  
High Affinity ◽  
Absolute Requirement ◽  
Potential Sensitivity ◽  
In Cells

Epithelial cells isolated from chick small intestine were used to study the mechanism of L-aspartate transport. Two kinetically distinct uptake systems of high (Km' = 16 microM) and low (Km'' = 2.7 mM) affinity are observed. This paper examines the cation dependence and membrane potential sensitivity of the high affinity system. Unidirectional influx studies indicate that extracellular Na+ is an absolute requirement for transport function. Flux is optimal when K+ is present intracellularly, however this cation is not required for Na+-dependent L-aspartate uptake. In the absence of K+, flux enhancement is observed when the intracellular pH is acidic. In contrast, acidic intracellular pH is inhibitory in cells that are preequilibrated with K+. Sodium ([Na+]o greater than [Na+]i gradients, and potassium ([K+]o less than [K+]i) or proton ([H+]o less than [H+]i) gradients can independently energize the Na+-dependent accumulation of L-aspartate above equilibrium levels, suggesting that Na+ and L-aspartate cotransport occurs with concomitant K+ or H+ antiport. L-Aspartate influx is insensitive to membrane potential changes created by inwardly directed anion gradients in the presence or absence of intracellular K+. A model is presented that is consistent with electroneutral Na+-coupled transfer with an ion antiport site of low specificity.

scholarly journals Understanding the effect of controlling phosphorothioate chirality in the DNA gap on the potency and safety of gapmer antisense oligonucleotides

2020 ◽  
Vol 48 (4) ◽  
pp. 1691-1700 ◽  
Author(s):  
Michael E Østergaard ◽  
Cheryl L De Hoyos ◽  
W Brad Wan ◽  
Wen Shen ◽  
Audrey Low ◽  
...  
Keyword(s):  
Antisense Oligonucleotides ◽  
Therapeutic Index ◽  
Rna Cleavage ◽  
Systematic Analysis ◽  
Phosphodiester Backbone ◽  
High Affinity ◽  
Backbone Modification ◽  
Therapeutic Profile ◽  
In Cells

Abstract Therapeutic oligonucleotides are often modified using the phosphorothioate (PS) backbone modification which enhances stability from nuclease mediated degradation. However, substituting oxygen in the phosphodiester backbone with sulfur introduce chirality into the backbone such that a full PS 16-mer oligonucleotide is comprised of 215 distinct stereoisomers. As a result, the role of PS chirality on the performance of antisense oligonucleotides (ASOs) has been a subject of debate for over two decades. We carried out a systematic analysis to determine if controlling PS chirality in the DNA gap region can enhance the potency and safety of gapmer ASOs modified with high-affinity constrained Ethyl (cEt) nucleotides in the flanks. As part of this effort, we examined the effect of systematically controlling PS chirality on RNase H1 cleavage patterns, protein mislocalization phenotypes, activity and toxicity in cells and in mice. We found that while controlling PS chirality can dramatically modulate interactions with RNase H1 as evidenced by changes in RNA cleavage patterns, these were insufficient to improve the overall therapeutic profile. We also found that controlling PS chirality of only two PS linkages in the DNA gap was sufficient to modulate RNase H1 cleavage patterns and combining these designs with simple modifications such as 2′-OMe to the DNA gap resulted in dramatic improvements in therapeutic index. However, we were unable to demonstrate improved potency relative to the stereorandom parent ASO or improved safety over the 2′-OMe gap-modified stereorandom parent ASO. Overall, our work shows that while controlling PS chirality can modulate RNase H1 cleavage patterns, ASO sequence and design are the primary drivers which determine the pharmacological and toxicological properties of gapmer ASOs.

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