Monitoring Real-Time Cyclic Nucleotide Dynamics in Subcellular Microdomains

2015 ◽  
pp. 152-163
Keyword(s):  
Real Time ◽  
Cyclic Nucleotide

scholarly journals Real-time visualization of conformational changes within single MloK1 cyclic nucleotide-modulated channels

2016 ◽  
Vol 7 (1) ◽  
Author(s):  
Martina Rangl ◽  
Atsushi Miyagi ◽  
Julia Kowal ◽  
Henning Stahlberg ◽  
Crina M. Nimigean ◽  
...  
Keyword(s):  
Real Time ◽  
Conformational Changes ◽  
Cyclic Nucleotide ◽  
High Speed ◽  
Function Analysis ◽  
Stable Conformation ◽  
Mesorhizobium Loti ◽  
Force Microscopy ◽  
Atomic Force ◽  
Real Time Visualization

AbstractEukaryotic cyclic nucleotide-modulated (CNM) ion channels perform various physiological roles by opening in response to cyclic nucleotides binding to a specialized cyclic nucleotide-binding domain. Despite progress in structure-function analysis, the conformational rearrangements underlying the gating of these channels are still unknown. Here, we image ligand-induced conformational changes in single CNM channels from Mesorhizobium loti (MloK1) in real-time, using high-speed atomic force microscopy. In the presence of cAMP, most channels are in a stable conformation, but a few molecules dynamically switch back and forth (blink) between at least two conformations with different heights. Upon cAMP depletion, more channels start blinking, with blinking heights increasing over time, suggestive of slow, progressive loss of ligands from the tetramer. We propose that during gating, MloK1 transitions from a set of mobile conformations in the absence to a stable conformation in the presence of ligand and that these conformations are central for gating the pore.

scholarly journals Role of 2′,3′-cyclic nucleotide 3′-phosphodiesterase in the renal 2′,3′-cAMP-adenosine pathway

2014 ◽  
Vol 307 (1) ◽  
pp. F14-F24 ◽  
Author(s):  
Edwin K. Jackson ◽  
Delbert G. Gillespie ◽  
Zaichuan Mi ◽  
Dongmei Cheng ◽  
Rashmi Bansal ◽  
...  
Keyword(s):  
Smooth Muscle ◽  
Vascular Smooth Muscle ◽  
Smooth Muscle Cells ◽  
Real Time ◽  
Vascular Smooth Muscle Cells ◽  
Real Time Pcr ◽  
Cyclic Nucleotide ◽  
Kidney Tissue ◽  
Energy Depletion ◽  
Proximal Tubular

Energy depletion increases the renal production of 2′,3′-cAMP (a positional isomer of 3′,5′-cAMP that opens mitochondrial permeability transition pores) and 2′,3′-cAMP is converted to 2′-AMP and 3′-AMP, which in turn are metabolized to adenosine. Because the enzymes involved in this “2′,3′-cAMP-adenosine pathway” are unknown, we examined whether 2′,3′-cyclic nucleotide 3′-phosphodiesterase (CNPase) participates in the renal metabolism of 2′,3′-cAMP. Western blotting and real-time PCR demonstrated expression of CNPase in rat glomerular mesangial, preglomerular vascular smooth muscle and endothelial, proximal tubular, thick ascending limb and collecting duct cells. Real-time PCR established the expression of CNPase in human glomerular mesangial, proximal tubular and vascular smooth muscle cells; and the level of expression of CNPase was greater than that for phosphodiesterase 4 (major enzyme for the metabolism of 3′,5′-cAMP). Overexpression of CNPase in rat preglomerular vascular smooth muscle cells increased the metabolism of exogenous 2′,3′-cAMP to 2′-AMP. Infusions of 2′,3′-cAMP into isolated CNPase wild-type (+/+) kidneys increased renal venous 2′-AMP, and this response was diminished by 63% in CNPase knockout (−/−) kidneys, whereas the conversion of 3′,5′-cAMP to 5′-AMP was similar in CNPase +/+ vs. −/− kidneys. In CNPase +/+ kidneys, energy depletion (metabolic poisons) increased kidney tissue levels of adenosine and its metabolites (inosine, hypoxanthine, xanthine, and uric acid) without accumulation of 2′,3′-cAMP. In contrast, in CNPase −/− kidneys, energy depletion increased kidney tissue levels of 2′,3′-cAMP and abolished the increase in adenosine and its metabolites. In conclusion, kidneys express CNPase, and renal CNPase mediates in part the renal 2′,3′-cAMP-adenosine pathway.

Real-Time Monitoring of Cyclic Nucleotide Changes in Living Cells

2019 ◽  
pp. 1-17
Author(s):  
Aniella Abi-Gerges ◽  
Khalil N. Khalil ◽  
Yara R. Neaimeh ◽  
Rodolphe Fischmeister
Keyword(s):  
Real Time ◽  
Cyclic Nucleotide ◽  
Living Cells ◽  
Real Time Monitoring

Some Recent Results in Quiescent Prominence Spectrometry

1979 ◽  
Vol 44 ◽  
pp. 41-47
Author(s):  
Donald A. Landman
Keyword(s):  
Real Time ◽  
Computer System ◽  
Spectral Response ◽  
Absolute Intensity ◽  
Solar Observatory ◽  
Target Size ◽  
Small Target ◽  
Signal Averaging ◽  
Quiescent Prominence

This paper describes some recent results of our quiescent prominence spectrometry program at the Mees Solar Observatory on Haleakala. The observations were made with the 25 cm coronagraph/coudé spectrograph system using a silicon vidicon detector. This detector consists of 500 contiguous channels covering approximately 6 or 80 Å, depending on the grating used. The instrument is interfaced to the Observatory’s PDP 11/45 computer system, and has the important advantages of wide spectral response, linearity and signal-averaging with real-time display. Its principal drawback is the relatively small target size. For the present work, the aperture was about 3″ × 5″. Absolute intensity calibrations were made by measuring quiet regions near sun center.

Video real-time imaging of artificial membranes during freeze-drying by cryo-electron microscopy

1988 ◽  
Vol 46 ◽  
pp. 16-17
Author(s):  
Alan S. Rudolph ◽  
Ronald R. Price
Keyword(s):  
Real Time ◽  
Self Assembly ◽  
Freeze Drying ◽  
Video Capture ◽  
Drying Process ◽  
Artificial Membranes ◽  
The Past ◽  
Cryo Electron Microscopy ◽  
Spherical Structures ◽  
Capture Techniques

We have employed cryoelectron microscopy to visualize events that occur during the freeze-drying of artificial membranes by employing real time video capture techniques. Artificial membranes or liposomes which are spherical structures within internal aqueous space are stabilized by water which provides the driving force for spontaneous self-assembly of these structures. Previous assays of damage to these structures which are induced by freeze drying reveal that the two principal deleterious events that occur are 1) fusion of liposomes and 2) leakage of contents trapped within the liposome [1]. In the past the only way to access these events was to examine the liposomes following the dehydration event. This technique allows the event to be monitored in real time as the liposomes destabilize and as water is sublimed at cryo temperatures in the vacuum of the microscope. The method by which liposomes are compromised by freeze-drying are largely unknown. This technique has shown that cryo-protectants such as glycerol and carbohydrates are able to maintain liposomal structure throughout the drying process.

An Interactive Minicomputer Program for the Indexing and Simulation of Electron Diffraction Patterns

1976 ◽  
Vol 34 ◽  
pp. 542-543
Author(s):  
R.P. Goehner ◽  
W.T. Hatfield ◽  
Prakash Rao
Keyword(s):  
Electron Diffraction ◽  
Real Time ◽  
Pattern Analysis ◽  
Flow Diagram ◽  
Hard Copy ◽  
Time Analysis ◽  
Real Time Analysis ◽  
Transmission Electron ◽  
One Step ◽  
Diffraction Patterns

Computer programs are now available in various laboratories for the indexing and simulation of transmission electron diffraction patterns. Although these programs address themselves to the solution of various aspects of the indexing and simulation process, the ultimate goal is to perform real time diffraction pattern analysis directly off of the imaging screen of the transmission electron microscope. The program to be described in this paper represents one step prior to real time analysis. It involves the combination of two programs, described in an earlier paper(l), into a single program for use on an interactive basis with a minicomputer. In our case, the minicomputer is an INTERDATA 70 equipped with a Tektronix 4010-1 graphical display terminal and hard copy unit.A simplified flow diagram of the combined program, written in Fortran IV, is shown in Figure 1. It consists of two programs INDEX and TEDP which index and simulate electron diffraction patterns respectively. The user has the option of choosing either the indexing or simulating aspects of the combined program.

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