Giuseppe Digilio

Iopamidol as a responsive MRI-chemical exchange saturation transfer contrast agent for pH mapping of kidneys: In vivo studies in mice at 7 T.

Iopamidol (Isovue®—Bracco Diagnostic Inc.) is a clinically approved X‐Ray contrast agent used in the last 30 years for a wide variety of diagnostic applications with a very good clinical acceptance. Iopamidol contains two types of amide functionalities that can be exploited for the generation of chemical exchange saturation transfer effect. The exchange rate of the two amide proton pools is markedly pH‐dependent. Thus, a ratiometric method for pH assessment has been set‐up based on the comparison of the saturation transfer effects induced by selective irradiation of the two resonances. This ratiometric approach allows to rule out the concentration effect of the contrast agent and provides accurate pH measurements in the 5.5–7.4 range. Upon injection of Iopamidol into healthy mice, it has been possible to acquire pH maps of kidney regions. Furthermore, it has been also shown that the proposed method is able to report about pH‐changes induced in control mice fed with acidified or basified water for a period of a week before image acquisition. Magn Reson Med, 2010.

Ubiad1 Is an Antioxidant Enzyme that Regulates eNOS Activity by CoQ10 Synthesis

Summary Protection against oxidative damage caused by excessive reactive oxygen species (ROS) by an antioxidant network is essential for the health of tissues, especially in the cardiovascular system. Here, we identified a gene with important antioxidant features by analyzing a null allele of zebrafish ubiad1, called barolo (bar). bar mutants show specific cardiovascular failure due to oxidative stress and ROS-mediated cellular damage. Human UBIAD1 is a nonmitochondrial prenyltransferase that synthesizes CoQ10 in the Golgi membrane compartment. Loss of UBIAD1 reduces the cytosolic pool of the antioxidant CoQ10 and leads to ROS-mediated lipid peroxidation in vascular cells. Surprisingly, inhibition of eNOS prevents Ubiad1-dependent cardiovascular oxidative damage, suggesting a crucial role for this enzyme and nonmitochondrial CoQ10 in NO signaling. These findings identify UBIAD1 as a nonmitochondrial CoQ10-forming enzyme with specific cardiovascular protective function via the modulation of eNOS activity.

Contrast agents for magnetic resonance angiographic applications: 1H and 17O NMR relaxometric investigations on two gadolinium(III) DTPA-like chelates endowed with high binding affinity to human serum albumin

N,N′,N″,N‴-pentaacetic acid) bearing different substituents for binding to human serum albumin (HSA) are compared. In spite of the structural differences of the recognition synthon and of the residual electric charge, the two chelates display an analogous binding affinity for the serum protein. Upon formation of the adducts with HSA, the exchange rates of the coordinated water appear slowed down by an amount corresponding to ca. 50% of the rates found for the free complexes. The relaxivity of [Gd(BOM)3DTPA (H2O)]2 −  is significantly higher than that of MS-325 either in the free complex or in the macromolecular adduct. Finally, the effect of pH on the stability of the HSA adducts and on the values of their relaxivities has been investigated.

EPR investigations of the iron domain in neuromelanin

The interactions between iron and neuromelanin (NM) have been studied by means of EPR spectroscopy. The variable temperature EPR spectral features of a specimen of NM extracted from normal human midbrains clearly indicate that iron is present as polynuclear oxy-hydroxy ferric aggregates as well as isolated Fe(III) centres. Ferric oxy-hydroxy phases are typical of the iron storage proteins ferritin and hemosiderin, but the comparison of the variable temperature EPR spectra of ferritin and NM highlights significant differences between the two iron(III)oxy-hydroxy domains. Moreover, further investigations on melanin models synthesised in the presence of either ferritin or a ferric salt as iron sources suggest that the same pathway of formation and inclusion of the polynuclear iron oxide is operating in NM and in the model systems, whatever is the source of iron.

Q-band EPR investigations of neuromelanin in control and Parkinson's disease patients

New insights into the understanding of the changes induced in the iron domain of neuromelanin (NM) upon development of Parkinsons disease (PD) have been gained by electron paramagnetic spectroscopy (EPR). The results of this study are compared with a previously reported variable temperature analysis of X-band EPR spectra of a NM specimen obtained from control brain tissues. The availability of high sensitivity instruments operating in the Q-band (34.4 GHz) allows us to deal with the low amounts of NM available from PD brains. The organization of iron in NM is in the form of polynuclear superparamagnetic/antiferromagnetic aggregates, but the lack of one or more signals in the EPR spectra of NM from PD suggests that the development of the pathology causes NM to decrease its ability to bind iron. Furthermore, the detection of the Mn(II) signal in the Q-band spectra is exploited as an additional internal probe to assess minor structural differences in iron domains of PD and control NM specimens.

Isolation and 13C-NMR characterization of an insoluble proteinaceous fraction from substantia nigra of patients with Parkinson's disease

Neuromelanin is a dark brown pigment suspected of being involved in the pathogenesis of Parkinsons disease. This pigment can be isolated from normal human substantia nigra by a procedure that includes an extensive proteolytic treatment. In this study we used such a procedure to extract the neuromelanin pigment from a pool of substantia nigra from patients affected by Parkinsons disease. 13C Cross polarization magic angle spinning nuclear magnetic resonance spectroscopy and electron paramagnetic resonance spectroscopy were used to characterize the solid residue obtained from the extraction procedure. We found that the pigment extracted from the substantia nigra of parkinsonian patients was mainly composed of highly cross‐linked, protease‐resistant, lipo‐proteic material, whereas the neuromelanin macromolecule appears to be only a minor component of this extract. A synthetic model of melanoprotein has been prepared by enzymatic oxidation of dopamine in the presence of albumin. Once it has undergone the same proteolytic treatment, this model system yields a 13C‐NMR spectrum which is similar to that observed for the parkinsonian midbrain extract. These results are consistent with the view that oxidative stress has a relevant role in the pathogenesis of Parkinsons disease.

Exofacial Protein Thiols as a Route for the Internalization of Gd(III)-Based Complexes for Magnetic Resonance Imaging Cell Labeling

Four novel MRI Gd(III)-based probes have been synthesized and evaluated for their labeling properties on cultured cell lines K562, C6, and B16. The labeling strategy relies upon the fact that cells display a large number of reactive exofacial protein thiols (EPTs) that can be exploited as anchorage points for suitably activated MRI probes. The probes are composed of a Gd(III) chelate (based on either DO3A or DTPA) connected through a flexible linker to the 2-pyridyldithio chemical function for binding to EPTs. GdDO3A-based chelates could efficiently label cells (up to a level of 1.2 x 10(10) Gd(III) atoms/cell), whereas GdDTPA-based chelates showed poor or no cell labeling ability at all. Among the GdDO3A based compounds, that having the longest spacer (compound GdL1A) showed the best labeling efficacy. The mechanism of EPT mediated cell labeling by GdL1A involves probe internalization without sequestration of the Gd(III) chelate within subcellular structures such as endosomes.

Nuclear magnetic relaxation dispersion profiles of substantia nigra pars compacta in Parkinson's disease patients are consistent with protein aggregation

Nuclear Magnetic Relaxation field-cycling relaxometry is a technique, able to report on water mobility in tissues. By means of this technique, post-mortem specimens from both controls and idiopathic Parkinsons disease patients have been investigated. Results show different relaxometric behavior between the groups, which is consistent with protein aggregation in Parkinsons disease specimens.

On the Fate of MRI Gd-Based Contrast Agents in Cells. Evidence for Extensive Degradation of Linear Complexes upon Endosomal Internalization

Commercial Gd-containing complexes are often used as MRI reporters in cellular labeling procedures as they are internalized into endosomes by pinocytosis. A methodology has been applied to assess the relative stability of three commercial Gd contrast agents following cellular uptake in fibroblasts and macrophages. It has been found that the acyclic series of Gd MRI contrast agents are degraded much more rapidly than their macrocyclic analogues, following endosomal internalization into living cells. This helps to explain their causal role in the development of nephrogenic systemic fibrosis in renally impaired patients. The methodology has also been applied to assess the fate of Gd-DTPA-BMA-loaded liposomes upon their endosomal internalization. Resistant liposomes prevent the degradation of the complex, whereas liposomes designed to release their payload in the acidic environments show a loss of integrity of Gd-DTPA-BMA analogous to the one observed upon internalization of the free complex.

Characterization of human hair melanin and its degradation products by means of magnetic resonance techniques

Melanin granules (MGs) have been extracted from human Chinese black hairs by either acid hydrolysis (CH‐type MGs) or enzymatic digestion (CP‐type MGs), and their chemical structure investigated at the solid state by means of 13C cross polarization magic angle spinning (CPMAS NMR) and EPR spectroscopy. Both types of MGs contain a large amount of protein that is tightly bound to the true melanin polymer, with CP‐type MGs having a larger protein content than CH‐type ones. Moreover, MGs may also contain variable amounts of lipid‐like material. A high amount of paramagnetic metals is detected by EPR in CP‐type MGs, in particular Fe(III). Iron can be bound in two chemical forms: as isolated high spin Fe(III) ions with rhombic symmetry and as small oxy‐hydroxy Fe(III) aggregates. Iron is poorly available to chelators. CH‐type MGs contain much fewer metals. CP‐type MGs have then been subjected to partial bleaching by hydrogen peroxide in ammonia, yielding a residual solid, called residual oxidized melanin (ROM) and a soluble but still pigmented fraction called melanin free acid (MFA). MFA can be isolated by precipitation at acidic pH. The 13C‐CPMAS NMR and EPR spectra of these derivatives indicated that ROM has a structure very similar to that of parent MGs, whereas MFA shows a decrease of the protein content with respect to the melanin and a decreased amount of bound iron. Thus, the oxidative degradation of CP‐type MGs is a process not involving the bulk of MGs, but rather it proceeds from the solvent‐exposed outer parts to the interior. Copyright