Direct Observation of Enhanced Nitric Oxide in a Murine Model of Diabetic Nephropathy.

Uncoupling of nitric oxide synthase (NOS) secondary to redox signaling is a central mechanism in endothelial and macrophage activation. To date studies on the production of nitric oxide (NO) during the development of diabetic complications show paradoxical results. We previously showed that recoupling eNOS by increasing the eNOS cofactor tetrahydrobiopterin (BH4) could restore endothelial function and prevent kidney injury in experimental kidney transplantation. Here, we employed a diabetic mouse model to investigate the effects of diabetes on renal tissue NO bioavailability. For this, we used in vivo NO trapping, followed by electron paramagnetic resonance spectroscopy. In addition, we investigated whether coupling of NOS by supplying the cofactor BH4 could restore glomerular endothelial barrier function. Our data show that overall NO availability at the tissue level is not reduced sixteen weeks after the induction of diabetes in apoE knockout mice, despite the presence of factors that cause endothelial dysfunction, and the presence of the endogenous NOS inhibitor ADMA. Targeting uncoupled NOS with the BH4 precursor sepiapterin further increases NO availability, but did not modify renal glomerular injury. Notably, glomerular heparanase activity as a driver for loss of glomerular barrier function was not reduced, pointing towards NOS-independent mechanisms. This was confirmed by unaltered increased glomerular presence of cathepsin L, the protease that activates heparanase.

Identification of free nitric oxide radicals in rat bone marrow: implications for progenitor cell mobilization in hypertension.

Nitric oxide (NO) has been implicated in matrix metallopeptidase 9 (MMP9)-dependent mobilization of hematopoietic stem and progenitor cells from bone marrow (BM). However, direct measurement of NO in the BM remained elusive due to its low in situ concentration and short lifetime. Using NO spin trapping and electron paramagnetic resonance (EPR) spectroscopy we give the first experimental confirmation of free NO radicals in rodent BM. NO production was quantified and attributed to enzymatic activity of NO synthases (NOS). Although endothelial NOS (eNOS) accounts for most (66%) of basal NO, we identified a significant contribution (23%) from inducible NOS (iNOS). Basal NO levels closely correlate with MMP9 bioavailability in BM of both hypertensive and control rats. Our observations support the hypothesis that inadequate mobilization of BM-derived stem and progenitor cells in hypertension results from impaired NOS/NO/MMP9 signalling in BM, a condition that may be corrected with pharmacological intervention.

NO formation by neuronal NO-synthase can be controlled by ultrafast electron injection from a nanotrigger.

Nitric oxide synthases (NOSs) are unique flavohemoproteins with various roles in mammalian physiology. Constitutive NOS catalysis is initiated by fast hydride transfer from NADPH, followed by slower structural rearrangements. We used a photoactive nanotrigger (NT) to study the initial electron transfer to FAD in native neuronal NOS (nNOS) catalysis. Molecular modeling and fluorescence spectroscopy showed that selective NT binding to NADPH sites close to FAD is able to override Phe1395 regulation. Ultrafast injection of electrons into the protein electron pathway by NT photoactivation through the use of a femtosecond laser pulse is thus possible. We show that calmodulin, required for NO synthesis by constitutive NOS, strongly promotes intramolecular electron flow (6.2-fold stimulation) by a mechanism involving proton transfer to the reduced FAD(-) site. Site-directed mutagenesis using the S1176A and S1176T mutants of nNOS supports this hypothesis. The NT synchronized the initiation of flavoenzyme catalysis, leading to the formation of NO, as detected by EPR. This NT is thus promising for time-resolved X-ray and other cellular applications.

Magnetic resonance imaging of phase transitions in nitinol.

Magnetic resonance images are prone to artifacts caused by metallic objects. Apart from being a source of image degradation, such artifacts can also provide information about the magnetic properties of the foreign object. In this work, we aim to explore the potential of magnetic resonance imaging to detect and characterize changes in magnetic properties of nitinol undergoing temperature- or strain-induced phase changes. A spin echo and a gradient echo method were used to measure the magnetization changes related to the phase transformations. Results of both methods were in agreement and in accordance with the independent measurements using a vibrating sample magnetometer. Magnetic resonance imaging turned out to be a suitable method to visualize and quantify magnetization and phase changes in situ. It is not restricted to a single imaging strategy and does not require any modification of the test object. The results indicate the potential of magnetic resonance imaging to provide direct feedback of the thermomechanical state of the alloy.

Magnetic resonance imaging of microstructure transition in stainless steel.

Magnetic resonance images are prone to artifacts caused by metallic objects. Such artifacts may not only hamper image interpretation, but also have been shown to provide information about the magnetic properties of the substances involved. In this work, we aim to explore the potential of MRI to detect, localize and characterize changes in magnetic properties that may occur when certain alloys have been exposed to a thermomechanical stress. For this purpose, stainless steel 304 L wires were drawn to induce a change from paramagnetic austenitic into ferromagnetic martensitic microstructure. The changes in magnetic behavior were quantified by analyzing the geometric distortion in spin echo and the geometric distortion and intravoxel dephasing in gradient echo images at 0.5, 1.5 and 3 T. The results of both imaging strategies were in agreement and in accordance with independent measurements with a vibrating sample magnetometer. Drawing wire to 2% of its cross-sectional area was found to increase the volume fraction of the ferromagnetic martensite from 0.3% to 80% and to enhance the magnetization up to two or three orders of magnitude. The results demonstrate the potential of MRI to locate and quantify stress-induced changes in the magnetic properties of alloys in a completely noninvasive and nondestructive way.