Acetoxymethyl Research Articles

Disulfide bonds reduce the toxicity of the amyloid fibrils formed by an extracellular protein.

Disulfide bonds reduce the toxicity of the amyloid fibrils formed by an extracellular protein.

Epac-Rap Signaling Reduces Cellular Stress and Ischemia-induced Kidney Failure

Renal ischemia-reperfusion injury is associated with the loss of tubular epithelial cell-cell and cell-matrix interactions which contribute to renal failure. The Epac-Rap signaling pathway is a potent regulator of cell-cell and cell-matrix adhesion. The cyclic AMP analogue 8-pCPT-2'-O-Me-cAMP has been shown to selectively activate Epac, whereas the addition of an acetoxymethyl (AM) ester to 8-pCPT-2'-O-Me-cAMP enhanced in vitro cellular uptake. Here we demonstrate that pharmacological activation of Epac-Rap signaling using acetoxymethyl-8-pCPT-2'-O-Me-cAMP preserves cell adhesions during hypoxia in vitro, maintaining the barrier function of the epithelial monolayer. Intrarenal administration in vivo of 8-pCPT-2'-O-Me-cAMP also reduced renal failure in a mouse model for ischemia-reperfusion injury. This was accompanied by decreased expression of the tubular cell stress marker clusterin-α, and lateral expression of β-catenin after ischemia indicative of sustained tubular barrier function. Our study emphasizes the undervalued importance of maintaining tubular epithelial cell adhesion in renal ischemia and demonstrates the potential of pharmacological modulation of cell adhesion as a new therapeutic strategy to reduce the extent of injury in kidney disease and transplantation.

A Comparative Assessment of Fluo Ca 2+ Indicators in Rat Ventricular Myocytes

Improvements in the fluo series of fluorescent Ca2+ indictors routinely used to measure cytosolic free Ca2+ concentration ([Ca2+]i) have increased signal-to-noise ratio, enabling more quantitative [Ca2+] measurements. The improved fluo derivatives show increased cellular loading efficiency, reduced pH sensitivity, and excitation maxima that better match the wavelengths of common lasers. Nevertheless, the extent to which these indicators interfere with native intracellular Ca2+ homeostasis has not been systematically characterized. Here, we have examined three different fluo derivatives (fluo-2, fluo-3 and fluo-4) in freshly isolated rat ventricular myocytes. Cells were loaded with a fluo indicator either by incubation with the acetoxymethyl (AM) ester or by introducing the K+ salt of the indicator through a whole-cell pipette (injection-loaded). Fluorescence changes in cardiomyocytes were measured using confocal microscopy during field simulation or current injection. Three significant differences were identified among the three indicators and two loading methods. 1. Ca2+ kinetics in AM-loaded cells were slower than in injection-loaded cells; thus the decay of the cardiac [Ca2+]i transient appeared to be slower. In AM-loaded cells, fluo-3 reported the fastest response while fluo-2 and fluo-4 gave comparable, slower responses. When injection-loaded as the K+ salt, all three indicators reported comparable responses that were faster than any of the AM-loaded indicators. 2. When AM-loaded, all three indicators showed apparent spatial inhomogeneities in cellular fluorescence, in contrast to injection-loaded cells. Thus diverse cellular structures appear brighter in AM-loaded cells than in injection-loaded cells. 3. For each indicator, the calibrated signals (F/Fo) in the AM-loaded cells were higher than in injection-loaded cells. We conclude that injection-loaded cells produce more accurate [Ca2+]i measurements and spatially resolved signals. If AM loading is needed, the use of fluo-3 would appear to provide more accurate calibrated signals.

Synthesis and utility of fluorogenic acetoxymethyl ethers.

Phenolic fluorophores such as fluorescein, Tokyo Green, resorufin, and their derivatives are workhorses of biological science. Acylating the phenolic hydroxyl group(s) in these fluorophores masks their fluorescence. The ensuing ester is a substrate for cellular esterases, which can restore fluorescence. These esters are, however, notoriously unstable to hydrolysis, severely compromising their utility. The acetoxymethyl (AM) group is an esterase-sensitive motif that can mask polar functionalities in small molecules. Here, we report on the use of AM ether groups to mask phenolic fluorophores. The resulting profluorophores have a desirable combination of low background fluorescence, high chemical stability, and high enzymatic reactivity, both in vitro and in cellulo. These simple phenyl ether-based profluorophores could supplement or supplant the use of phenyl esters for imaging biochemical and biological systems.

Synthesis, Antimicrobial Evaluation, and Docking Studies of Novel 4‐Substituted Quinazoline Derivatives as DNA‐Gyrase Inhibitors

Quinazoline derivatives are reported to have anti-inflammatory, analgesic, antibacterial, and anticancer activities. The incorporation of "OCH₂CONH₂" (oxymethylcarbamide) at 4th position of the quinazoline ring was found to influence the biological activities of the molecules. With this rationale, some new oxadiazolyl methyloxy quinazolines, pyrazolyl acetoxy methyl quinazolines, triazolylmethyloxy quinazolines were synthesized from anthranilic acid through quinazolyl oxyacetylhydrazide intermediates. All the compounds were characterized by IR, ¹H-NMR, EI-MS, and C, H, N analyses and evaluated for their antimicrobial activity. Docking studies on the DNA-gyrase enzyme (1KZN) show their role in the antimicrobial activity of the molecules and explain the higher potency of compounds 6a, 6b, 8a, 8b based on ReRanking scores and binding poses of the molecules.

ChemInform Abstract: Rearrangement of Unsaturated 2,4-O-Benzylidenehexitol Derivatives into C-Glycosylbenzene Derivatives.

Abstract Acetolysis of (Z)-1,3-di-O-acetyl-2,4-O-benzylidene-6-C-(2,4-dichlorophenyl)- d -xylo-hex-5-enitol (3) afforded (E)-1,2,3,4-tetra-O-acetyl-6-C-(2,4-dichlorophenyl)- d -xylo-hex-5-enitol and 2-C-[(R)-acetoxy (2,4-dichlorophenyl)methyl]-3,4,6-tri-O-acetyl-2-deoxy-β- l -galacto- and -β- l -gulo-hexopyranosylbenzene. The mechanism of this new rearrangement was studied by exchanging the substituents at C-1 and C-3 in 3 and those of the aromatic ring attached to C-6.

Techniques for Imaging Ca<sup>2+</sup> Signaling in Human Sperm

Fluorescence microscopy of cells loaded with fluorescent, Ca2+-sensitive dyes is used for measurement of spatial and temporal aspects of Ca2+ signaling in live cells. Here we describe the method used in our laboratories for loading suspensions of human sperm with Ca2+-reporting dyes and measuring the fluorescence signal during physiological stimulation. Motile cells are isolated by direct swim-up and incubated under capacitating conditions for 0-24 h, depending upon the experiment. The cell-permeant AM (acetoxy methyl ester) ester form of the Ca2+-reporting dye is then added to a cell aliquot and a period of 1 h is allowed for loading of the dye into the cytoplasm. We use visible wavelength dyes to minimize photo-damage to the cells, but this means that ratiometric recording is not possible. Advantages and disadvantages of this approach are discussed. During the loading period cells are introduced into an imaging chamber and allowed to adhere to a poly-D-lysine coated coverslip. At the end of the loading period excess dye and loose cells are removed by connection of the chamber to the perfusion apparatus. The chamber is perfused continuously, stimuli and modified salines are then added to the perfusion header. Experiments are recorded by time-lapse acquisition of fluorescence images and analyzed in detail offline, by manually drawing regions of interest. Data are normalized to pre-stimulus levels such that, for each cell (or part of a cell), a graph showing the Ca2+ response as % change in fluorescence is obtained.

Techniques for Imaging Ca<sup>2+</sup> Signaling in Human Sperm

Fluorescence microscopy of cells loaded with fluorescent, Ca2+-sensitive dyes is used for measurement of spatial and temporal aspects of Ca2+ signaling in live cells. Here we describe the method used in our laboratories for loading suspensions of human sperm with Ca2+-reporting dyes and measuring the fluorescence signal during physiological stimulation. Motile cells are isolated by direct swim-up and incubated under capacitating conditions for 0-24 h, depending upon the experiment. The cell-permeant AM (acetoxy methyl ester) ester form of the Ca2+-reporting dye is then added to a cell aliquot and a period of 1 h is allowed for loading of the dye into the cytoplasm. We use visible wavelength dyes to minimize photo-damage to the cells, but this means that ratiometric recording is not possible. Advantages and disadvantages of this approach are discussed. During the loading period cells are introduced into an imaging chamber and allowed to adhere to a poly-D-lysine coated coverslip. At the end of the loading period excess dye and loose cells are removed by connection of the chamber to the perfusion apparatus. The chamber is perfused continuously, stimuli and modified salines are then added to the perfusion header. Experiments are recorded by time-lapse acquisition of fluorescence images and analyzed in detail offline, by manually drawing regions of interest. Data are normalized to pre-stimulus levels such that, for each cell (or part of a cell), a graph showing the Ca2+ response as % change in fluorescence is obtained.

Imaging Action Potentials with Calcium Indicators: Figure 1.

The understanding of neuronal circuits has been, and will continue to be, greatly advanced by the simultaneous imaging of action potentials in neuronal ensembles. This protocol describes "bulk" loading of brain slices with acetoxymethyl (AM) ester calcium indicators in order to monitor action potential activity in large populations of neurons simultaneously. The imaging of calcium influx into neurons provides an indirect, but accurate, measure of action potential generation in individual neurons. Single-cell resolution, and thus the easy identification of every active cell, is the key advantage of the technique.

Thermoresponsive Polyphosphoesters Bearing Enzyme-cleavable Side Chains

Abstract Thermoresponsive polyphosphoesters bearing acetoxymethyl (AM) groups were newly synthesized. The polymers had the LCST-type cloud point in aqueous media, and the thermoresponsive phenomena was influenced by the condition of the medium. About 40% of the AM groups in the polymers were cleaved in contact with esterase for 24 h in phosphate buffer solution. This was significant compared with the spontaneous cleavage in the buffer solution. After the cleavage of the AM groups, the thermoresponsivity of the polyphosphoesters was diminished.

Studies on Triple Base Gun Propellant Based on Two Energetic Azido Esters

Triple base propellant is the workhorse propellant because it possesses several advantages like reduced flash, flame temperature, and erosion of the barrel as compared to double and single base propellant. Hence, efforts are going on worldwide to increase its performance by increasing its energy using energetic plasticizers and binders. In the present article, nonenergetic plasticizer dibutyl phthalate (DBP) is replaced by two energetic azido ester plasticizers, tris(azido acetoxy methyl) propane (TAAMP) and bis(azido acetoxy) bis(azido methyl) propane (BABAMP), in triple base composition and their different properties are studied. Experimental closed vessel (CV) results (loading density 0.2 g/cm3) clearly indicate that the triple base composition with 2% DBP has force constant 1018 J/g, which is increased to 1026 and 1030 J/g on replacement of DBP by 1 and 2% TAAMP, respectively. Mechanical properties of propellant compositions containing 1 and 2% TAAMP (compression strength 279 and 291 kgf/cm2, percentage compression 11.2 and 10.5, respectively) are also better than those of composition containing DBP (compression strength 275 kgf/cm2 and 10.3% compression). Similarly, 1 and 2% replacement of DBP by BABAMP shows further rise in energy (1032 J/g, 1038 J/g respectively) than that of compositions containing 1 and 2% TAAMP. These two compositions also exhibit better mechanical properties (compression strength 311.2 and 312.3 kgf/cm2, % compression 11.0 and 10.5, respectively) than compositions containing 1 and 2% TAAMP. The differential thermal analysis (DTA) results brought out the fact that the compositions containing energetic plasticizers revealed maximum decomposition temperature in the range of 172–174°C which is close to DBP plasticized triple base gun propellants (174°C). The energetic plasticized propellant compositions of both (TAAMP and BABAMP) showed sensitivity data in the range of (H50 19 to 22 cm, F of I 25 to 29 and friction insensitivity 19.2 kg) acceptable limit for gun propellant.

Highlighting Synaptic Communication in the Enteric Nervous System

Highlighting Synaptic Communication in the Enteric Nervous System

A new non-disruptive strategy to target calcium indicator dyes to the endoplasmic reticulum

For the analysis of Ca(2+)-dependent signaling, acetoxymethyl (AM)-derivatized ion indicators have become a popular tool. These indicators permeate membranes in an ion-insensitive form but, within cells, esterases hydrolyze these compounds to release ion-sensitive dyes. However, the properties of these indicators Limit their targeting to subcellular structures such as the endoplasmic reticulum, the dominant intracellular Ca2+ store. This study presents a novel approach for trapping fluorescent Ca2+ indicators in the ER. The method combines the selectivity of protein targeting with the biochemical advantages of synthetic Ca2+ indicators and allows direct, non-disruptive measurements of Ca(2+)-store dynamics with a high structural and temporal resolution. A recombinant carboxylesterase was targeted to the ER, providing a local esterase activity. After esterase-based dye loading, this additional esterase activity allowed improved trapping of Ca(2+)-sensitive forms of low-affinity Ca2+ indicators (e.g. Fluo5N) within the ER. The utility of the method was confirmed using different cell systems (293T, BHK21, cortical neurons) and activating different signaling pathways. In neurons, this approach enabled the detection of ER Ca2+ release with high resolution. In addition, the method allowed rapid confocal imaging of Ca2+ release from the ER, after activation of metabotropic glutamate receptors, in the presence of extracellular Ca2+.

Inhibition of Macrophage Phagocytotic Activity by a Receptor-targeted Polymer Vesicle-based Drug Delivery Formulation of Pravastatin

Ruptures of macrophage-rich atherosclerotic plaques in the coronary arteries are the main reason for heart attack. Targeted therapeutic interventions with an inhibitory effect on the macrophages promise to be beneficial, but currently available drugs such as statins achieve event reductions of only 30%. Dose-limiting adverse effects in remote organs prohibit achieving higher drug levels known to have strong inhibitory effects on macrophages. Receptor-specific targeting using statin-loaded nanometer-sized triblock copolymer vesicles with targeting moieties might allow high-dose treatment for improved efficacy, while minimizing toxicity in other cells. Vesicle uptake by target cells but not other cell types and slow intracellular content release was observed. A major improvement in biologic efficacy was observed for polymer vesicles compared to free drug, whereas no increased cytotoxicity was observed in muscle cells. Such high-dose, targeted therapy of statins through cell-specific polymer vesicles allows novel treatment paradigms not only for atherosclerosis, but appears promising for a wide range of drugs and diseases.

Controlled on-chip stimulation of quantal catecholamine release from chromaffin cells using photolysis of caged Ca2+on transparent indium-tin-oxide microchip electrodes

Photorelease of caged Ca(2+) is a uniquely powerful tool to study the dynamics of Ca(2+)-triggered exocytosis from individual cells. Using photolithography and other microfabrication techniques, we have developed transparent microchip devices to enable photorelease of caged Ca(2+), together with electrochemical detection of quantal catecholamine secretion from individual cells or cell arrays as a step towards developing high-throughput experimental devices. A 100 nm thick transparent indium-tin-oxide (ITO) film was sputter-deposited onto glass coverslips, which were then patterned into 24 cell-sized working electrodes (approximately 20 microm by 20 microm). We loaded bovine chromaffin cells with acetoxymethyl (AM) ester derivatives of the Ca(2+) cage NP-EGTA and Ca(2+) indicator dye fura-4F, then transferred these cells onto the working ITO electrodes for amperometric recordings. Upon flash photorelease of caged Ca(2+), a uniform rise of [Ca(2+)](i) within the target cell leads to quantal release of oxidizable catecholamines measured amperometrically by the underlying ITO electrode. We observed a burst of amperometric spikes upon rapid elevation of [Ca(2+)](i) and a "priming" effect of sub-stimulatory [Ca(2+)](i) on the response of cells to subsequent [Ca(2+)](i) elevation, similar to previous reports using different techniques. We conclude that UV photolysis of caged Ca(2+) is a suitable stimulation technique for higher-throughput studies of Ca(2+)-dependent exocytosis on transparent electrochemical microelectrode arrays.

Very‐low‐frequency electron paramagnetic resonance (EPR) imaging of nitroxide‐loaded cells

Recent advances in electron paramagnetic resonance (EPR) imaging have made it possible to image, in real time in vivo, cells that have been labeled with nitroxide spin probes. We previously reported that cells can be loaded to high (millimolar) intracellular concentrations with (2,2,5,5-tetramethylpyrrolidin-1-oxyl-3-ylmethyl)amine-N,N-diacetic acid by incubation with the corresponding acetoxymethyl (AM) ester. Furthermore, the intracellular lifetime (t(1/e)) of this nitroxide is 114 min-sufficiently long to permit in vivo imaging studies. In the present study, at a gradient of approximately 50 mT/m, we acquire and compare EPR images of a three-tube phantom, filled with either a 200-microM solution of the nitroxide, or a suspension of cells preincubated with the nitroxide AM ester. In both cases, 3-mm resolution images can be acquired with excellent signal-to-noise ratios (SNRs). These findings indicate that cells well-loaded with nitroxide are readily imageable by EPR imaging, and that in vivo tracking studies utilizing such cells should be feasible.

Membrane recycling and calcium dynamics during settlement and adhesion of zoospores of the green alga Ulva linza

Recruitment of individuals of the marine alga Ulva linza on to a suitable habitat involves the settlement of motile zoospores on to a substratum during which a preformed adhesive is secreted by vesicular exocytosis. The fluorescent styryl dye FM 1-43 and fluorescent Ca(2+) indicators were used to follow membrane cycling and changes in cytosolic Ca(2+) ([Ca(2+)](cyt)) associated with settlement. When swimming zoospores were exposed continuously to FM 1-43, the plasma membrane was preferentially labelled. During settlement, FM 1-43-labelled plasma membrane was rapidly internalized reflecting high membrane turnover. The internalized membrane was focused into a discrete region indicating targeting of membrane to an endosome-like compartment. Acetoxymethyl (AM)-ester derivatives were found to be unsuitable for monitoring [Ca(2+)](cyt) because the dyes were rapidly sequestered from the cytoplasm into sub-cellular compartments. [Ca(2+)](cyt) was, however, reliably measured using dextran-conjugated calcium indicators delivered into cells using a biolistic technique. Cells loaded with Oregon Green BAPTA-1 dextran (Invitrogen, Paisley, UK) showed diffuse cytosolic loading and reliably responded to imposed changes in [Ca(2+)](cyt). During settlement, zoospores exhibited both localized and diffuse increases in [Ca(2+)](cyt) implying a role for [Ca(2+)](cyt) in exocytosis of the adhesive.

Photochemical gating of intracellular Ca2+ release channels.

We have synthesized BiNiX, a caged methylxanthine agonist for the ryanodine receptor (RyR)the major calcium channel that mediates Ca2+ release from intracellular Ca2+ stores in electrically excitable cells. BiNiX is easily loaded into living cells through incubation with its acetoxymethyl (AM) ester. Delivery of focused UV light pulses to the loaded cell releases paraxanthine focally and rapidly to activate RyRs to release Ca2+, thus elevating intracellular Ca2+ concentration to initiate Ca2+-dependent signaling cascades. We demonstrate the biological utility of BiNiX in heart muscle cells by showing that local intracellular photolysis of BiNiX triggers Ca2+ release and consequent muscle contraction.

Sodium sensing induces different changes in free cytosolic calcium concentration and pH in salt‐tolerant and ‐sensitive rice (Oryza sativa) cultivars

Perception of salt stress in plant cells induces a change in the free cytosolic Ca2+, [Ca2+]cyt, which transfers downstream reactions toward salt tolerance. Changes in cytosolic H+ concentration, [H+]cyt, are closely linked to the [Ca2+]cyt dynamics under various stress signals. In this study, salt‐induced changes in [Ca2+]cyt, and [H+]cyt and vacuolar [H+] concentrations were monitored in single protoplasts of rice (Oryza sativa L. indica cvs. Pokkali and BRRI Dhan29) by fluorescence microscopy. Changes in cytosolic [Ca2+] and [H+] were detected by use of the fluorescent dyes acetoxy methyl ester of calcium‐binding benzofuran and acetoxy methyl ester of 2′, 7′‐bis‐(2‐carboxyethyl)‐5‐(and‐6) carboxyfluorescein, respectively, and for vacuolar pH, fluorescent 6‐carboxyfluorescein and confocal microscopy were used. Addition of NaCl induced a higher increase in [Ca2+]cyt in the salt‐tolerant cv. Pokkali than in the salt‐sensitive cv. BRRI Dhan29. From inhibitor studies, we conclude that the internal stores appear to be the major source for [Ca2+]cyt increase in Pokkali, although the apoplast is more important in BRRI Dhan29. The [Ca2+]cyt measurements in rice also suggest that Na+ should be sensed inside the cytosol, before any increase in [Ca2+]cyt occurs. Moreover, our results with individual mesophyll protoplasts suggest that ionic stress causes an increase in [Ca2+]cyt and that osmotic stress sharply decreases [Ca2+]cyt in rice. The [pH]cyt was differently shifted in the two rice cultivars in response to salt stress and may be coupled to different activities of the H+‐ATPases. The changes in vacuolar pH were correlated with the expressional analysis of rice vacuolar H+‐ATPase in these two rice cultivars.

Limited utility of acetoxymethyl (AM)‐based intracellular delivery systems, in vivo: interference by extracellular esterases

The use of acetoxymethyl (AM) groups to deliver and trap exogenous optical probes inside cells is an established tool in cell biology/physiology, however, these probes have not been used extensively in vivo. In this study, the use of the acetoxymethyl delivery system for optical probes was evaluated, in vivo. Initial studies revealed very little trapped probe in intact tissues even when near saturating levels of probe were injected in living animals. We tested the hypothesis that extracellular esterases rapidly cleave the acetoxymethyl groups preventing the probes from entering cells, in vivo. The rates of hydrolysis of 11 acetoxymethyl probes in diluted porcine plasma revealed an essentially first order high rate dye cleavage with half times on the order of minutes or less. Studies on mice and rabbits revealed rates 10- to 2-fold higher, respectively. These plasma studies suggested that the acetoxymethyl probes were being cleaved before having a chance to enter cells in tissues in vivo. This was confirmed using intravital 2-photon excitation microscopy in muscle tissue where several acetoxymethyl probes were found to rapidly cleave in the vascular space during infusion and not be trapped in the muscle cells. Studies with succinimidyl esters that should quickly bind to proteins on cleavage also failed to enter cells, in vivo, consistent with the notion that the cleavage was occurring in the extracellular space. These data suggest that the high level of plasma and extracellular esterase activity render the classical acetoxymethyl probes ineffective for monitoring intracellular events, in vivo. Different approaches to trapping exogenous probes will need to be explored for physiological studies using intravital microscopy.