Measuring Biochemical Reaction Rates In Vivo with Magnetization Transfer

2016 ◽  
pp. 843-858 ◽  
Author(s):  
Paul A. Bottomley
Keyword(s):  
Magnetization Transfer ◽  
Reaction Rates ◽  
Biochemical Reaction

scholarly journals mTORC1 controls phase-separation and the biophysical properties of the cytoplasm by tuning crowding

2017 ◽  
Author(s):  
M. Delarue ◽  
G.P. Brittingham ◽  
S. Pfeffer ◽  
I.V. Surovtsev ◽  
S. Ping-lay ◽  
...  
Keyword(s):  
Phase Separation ◽  
Fluorescent Protein ◽  
Effective Diffusion ◽  
Reaction Rates ◽  
Biophysical Properties ◽  
Cell Interior ◽  
Profound Impact ◽  
Mtorc1 Pathway

Summary (Abstract)Macromolecular crowding has a profound impact on reaction rates and the physical properties of the cell interior, but the mechanisms that regulate crowding are poorly understood. We developed Genetically Encoded Multimeric nanoparticles (GEMs) to dissect these mechanisms. GEMs are homomultimeric scaffolds fused to a fluorescent protein. GEMs self-assemble into bright, stable fluorescent particles of defined size and shape. By combining tracking of GEMs with genetic and pharmacological approaches, we discovered that the mTORC1 pathway can tune the effective diffusion coefficient of macromolecules ≥15 nm in diameter more than 2-fold without any discernable effect on the motion of molecules ≥5 nm. These mTORCI-dependent changes in crowding and rheology affect phase-separation both in vitro and in vivo. Together, these results establish a role for mTORCI in controlling both the biophysical properties of the cytoplasm and the phase-separation of biopolymers.

scholarly journals Fast Isotopic Exchange between Mitochondria and Cytosol in Brain Revealed by Relayed 13C Magnetization Transfer Spectroscopy

2009 ◽  
Vol 29 (4) ◽  
pp. 661-669 ◽  
Author(s):  
Jehoon Yang ◽  
Su Xu ◽  
Jun Shen
Keyword(s):  
Isotopic Exchange ◽  
Tricarboxylic Acid Cycle ◽  
Magnetization Transfer ◽  
Tricarboxylic Acid ◽  
Transfer Effect ◽  
Resonance Spectroscopy ◽  
Exchange Reactions ◽  
Acid Cycle ◽  
Longitudinal Relaxation

In vivo13C magnetic resonance spectroscopy has been applied to studying brain metabolic processes by measuring 13C label incorporation into cytosolic pools such as glutamate and aspartate. However, the rate of exchange between mitochondrial α-ketoglutarate/oxaloacetate and cytosolic glutamate/aspartate ( Vx) extracted from metabolic modeling has been controversial. Because brain fumarase is exclusively located in the mitochondria, and mitochondrial fumarate is connected to cytosolic aspartate through a chain of fast exchange reactions, it is possible to directly measure Vx from the four-carbon side of the tricarboxylic acid cycle by magnetization transfer. In isoflurane-anesthetized adult rat brain, a relayed 13C magnetization transfer effect on cytosolic aspartate C2 at 53.2ppm was detected after extensive signal averaging with fumarate C2 at 136.1ppm irradiated using selective radiofrequency pulses. Quantitative analysis using Bloch–McConnell equations and a four-site exchange model found that VxE13–19 µmol per g per min (≫ VTCA, the tricarboxylic acid cycle rate) when the longitudinal relaxation time of malate C2 was assumed to be within ±33% of that of aspartate C2. If VxE VTCA, the isotopic exchange between mitochondria and cytosol would be too slow on the time scale of 13C longitudinal relaxation to cause a detectable magnetization transfer effect.

scholarly journals Multiparametric MRI detects longitudinal evolution of folic acid-induced nephropathy in mice

2018 ◽  
Vol 315 (5) ◽  
pp. F1252-F1260 ◽  
Author(s):  
Kai Jiang ◽  
Tristan A. Ponzo ◽  
Hui Tang ◽  
Prasanna K. Mishra ◽  
Slobodan I. Macura ◽  
...  
Keyword(s):  
Folic Acid ◽  
Interstitial Fibrosis ◽  
Magnetization Transfer ◽  
Kidney Injury ◽  
Multiparametric Mri ◽  
Magnetization Transfer Ratio ◽  
Kidney Volume ◽  
Kidney Size ◽  
Functional Changes

The rodent model of folic acid (FA)-induced acute kidney injury (AKI) provides a useful model for studying human AKI, but little is known about longitudinal changes in renal hemodynamics and evolution of renal fibrosis in vivo. In this work, we aimed to longitudinally assess renal structural and functional changes using multiparametric magnetic resonance imaging (MRI). Ten adult mice were injected with FA, after which a multiparametric MRI was used to measure kidney volume, hypoxia index R2*, magnetization transfer ratio (MTR), perfusion, T1, and glomerular filtration rate (GFR) at 2 wk posttreatment. Then five mice were euthanized for histology, and the other five underwent MRI again at 4 wk, followed by histology. Control mice ( n = 5) were injected with vehicle and studied with MRI at 2 wk. Trichrome and hematoxylin-eosin staining were performed to assess FA-induced tissue injuries. Whereas kidney size and oxygenation showed progressive deterioration, a transient impairment in renal perfusion and normalized GFR slightly improved by 4 wk. Kidney fluid content, as reflected by T1, was prominent at 2 wk and tended to regress at 4 wk, consistent with observed tubular dilation. Trichrome staining revealed patchy necrosis and mild interstitial fibrosis at 2 wk, which exacerbated at 4 wk. MTR detected increased fibrosis at 4 wk. In conclusion, multiparametric MRI captured the longitudinal progression in kidney damage evolving within the first month after treatment with folic acid and may provide a useful tool for assessment of therapeutic strategies.

scholarly journals Molecular immunocytochemistry of the CuZn superoxide dismutase in rat hepatocytes.

1988 ◽  
Vol 107 (6) ◽  
pp. 2169-2179 ◽  
Author(s):  
L Y Chang ◽  
J W Slot ◽  
H J Geuze ◽  
J D Crapo
Keyword(s):  
Superoxide Dismutase ◽  
Rat Liver ◽  
Oxidant Stress ◽  
Rat Hepatocytes ◽  
Reaction Rates ◽  
Cytoplasmic Matrix ◽  
Cytosolic Proteins ◽  
Cuzn Superoxide Dismutase ◽  
O2 Production

The distribution of CuZn superoxide dismutase (SOD) molecules in subcellular organelles in rat liver hepatocytes was studied using integrated biochemical, stereological, and quantitative immunocytochemical techniques. A known concentration of purified CuZn SOD in 10% gelatin was embedded alongside the liver tissue for the calculation of CuZn SOD concentrations in hepatocyte organelles and total CuZn SOD in the rat liver. Most of the CuZn SOD was located in the cytoplasmic matrix (73.1%) and in the nucleus (11.9%) with concentrations of 1.36 and 0.71 mg/cm3, respectively. Lysosomes contained the highest concentration (5.81 mg/cm3). Only low concentrations were measured in mitochondria (0.21 mg/cm3). Membrane-bound spaces of rough endoplasmic reticulum (ER), smooth ER, and the Golgi system did not contain significant concentrations of the enzyme. By adding up the concentrations in all subcellular compartments, a total liver content of CuZn SOD was established from the immunocytochemical measurements (0.386 +/- 0.028 mg/gm liver) that agreed closely with those obtained by biochemical assays (0.380 +/- 0.058 mg/gm liver). The average distances between two CuZn SOD molecules can be calculated from enzyme concentrations. It was determined that CuZn SOD molecules in the cytoplasmic matrix and nucleus were 34 and 42 nm apart, respectively. In peroxisomes and mitochondria, average intermolecular distance increased to approximately 60 nm and increased to 136 nm in smooth ER. CuZn SOD is a relatively abundant protein in the cytosol of hepatocytes and its distribution overlaps with major sites of O2- production. The efficiency of protection CuZn SOD can provide to cytosolic proteins from attacks by superoxide anion depends on the rate of O2- production, distribution of CuZn SOD relative to cytosolic proteins, and the relative reaction rates between O2- with both cytosolic proteins and CuZn SOD. Future studies of these substrate-enzyme relationships in vivo can lead to a greater understanding of how cells handle oxidant stress.

Use of magnetization transfer imaging in vivo to investigate the neuropathology in schizophrenia

1998 ◽  
Vol 29 (1-2) ◽  
pp. 80 ◽  
Author(s):  
J. Foong ◽  
M. Maier ◽  
G. Barker ◽  
D.H. Miller ◽  
M.A. Ron
Keyword(s):  
Magnetization Transfer ◽  
Magnetization Transfer Imaging

NMR studies of enzymatic rates in vitro and in vivo by magnetization transfer

1984 ◽  
Vol 17 (1) ◽  
pp. 83-124 ◽  
Author(s):  
J. R. Alger ◽  
R. G. Shulman
Keyword(s):  
Creatine Kinase ◽  
Chemical Reactions ◽  
Magnetization Transfer ◽  
Living Systems ◽  
Nmr Studies ◽  
Wide Range ◽  
Potential Applications

Magnetization transfer techniques are specialized NMR experiments which can measure the rate of chemical reactions while concentrations of products and reactants are maintained constant. These techniques are being used to measure the rates of enzyme catalysed reactions in a variety of living systems and in vitro. The magnetization transfer measurements in vivo of the ATP synthetase and the creatine kinase reactions have been particularly useful in describing rates of major energy transducing reactions involving ATP and phosphocreatine. As a result, a wide range of biomedicai scientists are becoming aware of the potentials of these techniques. The purpose of this review is thus threefold: first, to present a concise, conceptual review of the underlying principles for these non-specialists; secondly, to review the important biochemical applications of the method which have appeared, and thirdly, to discuss potential applications and limitations of the method.

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