Biomedical subjects

M Fasano

Publications and source records attributed to M Fasano.

At least 19 recordsLinked to original sources

PubMed2006

Is neuromelanin changed in Parkinson's disease? Investigations by magnetic spectroscopies.

Human mesencephalic neuromelanin (NM) is characterized by an irregular, undefined structure, making its characterization by usual physico-chemical methodologies quite difficult. NM isolated from controls and from Parkinson's Disease (PD) patients was compared by high-resolution solid-state nuclear magnetic resonance (NMR). The pigment from PD patients appeared to be mainly composed of highly cross-linked, protease-resistant lipo-proteic material, with disappearance of melanin NMR resonances, suggesting melanin breakout due to oxidative stress conditions. Moreover, alpha-synuclein was detected in NM of PD patients and controls after cleavage of the melanin backbone under solubilizing conditions. NM stores iron ions as oxyhydroxide iron clusters containing thousands of iron atoms. Electron Paramagnetic Resonance (EPR) investigations and magnetic susceptibility measurements confirmed the occurrence of magnetic coupling among iron atoms, whereas in synthetic melanin the occurrence of isolated Fe(3+) ions was evident. NM from PD patients showed a lower total magnetization, possibly suggesting a progressive Fe migration from its storage environment (i.e., NM) to the cytosol.

Humans↗

PubMed2003

Proteome analysis of mesencephalic tissues: evidence for Parkinson's disease.

Proteome analysis is a powerful methodology to investigate protein expression in tissues involved in diseases not linked to particular genetic defects. To date, this technique has a limited number of applications in the field of neurodegenerative disorders. We decided therefore to investigate by this approach autoptic mesencephalic tissues of patients with idiopathic Parkinson's disease as well as control specimens from healthy subjects.

Blotting, Western↗

PubMed2002

Magnetic investigations of human mesencephalic neuromelanin.

Pigmentation of neurons in substantia nigra is due to neuromelanin, a pigment that stores large amounts of iron. Human mesencephalic neuromelanin has been investigated by means of magnetic susceptibility measurements as a function of temperature. Magnetic measurements provide a physico-chemical characterization of the iron cluster buried in the organic melanin matrix and support the view that iron is not simply chelated, but rather is organized in a three-dimensional network. The paramagnetism of isolated iron ions is observed, in agreement with electron paramagnetic resonance spectroscopy. Furthermore, antiferromagnetic grains with a large size distribution function are present. These grains contain N spins coupled antiferromagnetically; however, N(1/2) spins are decoupled from the grain bulk and parallelly aligned. The latter subgrains are superparamagnetic with a blocking temperature ranging between 5 K and room temperature. This behavior has not been observed in synthetic melanin, where the paramagnetic contribution is strongly enhanced. Preliminary results on pigment isolated from patients affected by Parkinson's disease, a neurodegenerative pathology involving primarily pigmented neurons in substantia nigra pars compacta, show a lower total magnetization compared to control neuromelanin. The temperature behavior of zero field cooling and field cooling magnetizations is similar for both. The significant depletion of iron content in Parkinson's disease neuromelanin could indicate a progressive Fe migration from its storage environment to the cytosol.

Ferric Compounds↗

PubMed2001

Relaxometric characterization of human hemalbumin.

Hemalbumin [i.e., Fe(III)-protoporphyrin IX-human serum albumin; Fe(III)heme-HSA] is an important intermediate in the recovery of heme iron following hemolysis. Relaxometric data are consistent with the occurrence of a hexacoordinated high-spin Fe(III) center with no water in the inner coordination sphere. The relatively high relaxation enhancement observed for an aqueous solution of Fe(III)heme-HSA (r1p=4.8 mM(-1)s(-1) at 20 MHz, pH 7, and 25 C) is ascribed to the occurrence of a strong contribution from water molecules in the second coordination sphere. Structural analysis of the putative binding region has been performed by a Monte Carlo simulated annealing procedure, which allowed us to identify His105 and Tyr148 as axial ligands. The role of a tyrosinate as the sixth Fe(III)heme ligand is supported by the pH-dependent analysis. Interestingly, when Fe(III) is replaced by Mn(III), the occurrence of a fast exchanging water molecule at pH values close to neutrality is detected. As the pH is increased, the Mn(III) containing system behaves analogously to Fe(III)heme-HSA. At higher pH, the phenolate ligand is eventually displaced by OH- from both Fe(III) and Mn(III) centers. Support for the proposed bonding scheme has been gained also from competitive binding assays for the sixth coordination site by fluoride, azide, and imidazole ligands.

Binding Sites↗

PubMed2001

Effect of bezafibrate and clofibrate on the heme-iron geometry of ferrous nitrosylated heme-human serum albumin: an EPR study.

The effect of bezafibrate (BZF) and clofibrate (CF), two therapeutic drugs displaying anticoagulant and antihyperlipoproteinemic activities, on the EPR-spectroscopic properties of ferrous nitrosylated heme-human serum albumin (HSA-heme-NO) has been investigated. In the absence of BZF and CF, HSA-heme-NO is a five-coordinate heme-iron system, characterised by an X-band EPR spectrum with a three-line splitting in the high magnetic field region. Addition of BZF and CF to HSA-heme-NO induced the transition towards a six-coordinate heme-iron species characterised by an X-band EPR spectrum with an axial geometry. These data indicate that HSA-heme-NO is a five-coordinate heme-iron system, BZF and CF acting as allosteric effectors, and show that the primary heme binding site and the CF cleft of HSA are conformationally-linked, regardless of their different location.

Bezafibrate↗

PubMed2001

Effect of ibuprofen and warfarin on the allosteric properties of haem-human serum albumin. A spectroscopic study.

Haem binding to human serum albumin (HSA) endows the protein with peculiar spectroscopic properties. Here, the effect of ibuprofen and warfarin on the spectroscopic properties of ferric haem-human serum albumin (ferric HSA-haem) and of ferrous nitrosylated haem-human serum albumin (ferrous HSA-haem-NO) is reported. Ferric HSA-haem is hexa-coordinated, the haem-iron atom being bonded to His105 and Tyr148. Upon drug binding to the warfarin primary site, the displacement of water molecules--buried in close proximity to the haem binding pocket--induces perturbation of the electronic absorbance properties of the chromophore without affecting the coordination number or the spin state of the haem-iron, and the quenching of the 1H-NMR relaxivity. Values of Kd for ibuprofen and warfarin binding to the warfarin primary site of ferric HSA-haem, corresponding to the ibuprofen secondary cleft, are 5.4 +/- 1.1 x 10(-4) m and 2.1 +/- 0.4 x 10(-5) m, respectively. The affinity of ibuprofen and warfarin for the warfarin primary cleft of ferric HSA-haem is lower than that reported for drug binding to haem-free HSA. Accordingly, the Kd value for haem binding to HSA increases from 1.3 +/- 0.2 x 10(-8) m in the absence of drugs to 1.5 +/- 0.2 x 10(-7) m in the presence of ibuprofen and warfarin. Ferrous HSA-haem-NO is a five-coordinated haem-iron system. Drug binding to the warfarin primary site of ferrous HSA-haem-NO induces the transition towards the six-coordinated haem-iron species, the haem-iron atom being bonded to His105. Remarkably, the ibuprofen primary cleft appears to be functionally and spectroscopically uncoupled from the haem site of HSA. Present results represent a clear-cut evidence for the drug-induced shift of allosteric equilibrium(a) of HSA.

Allosteric Site↗

PubMed2000

Investigation of the active site of Escherichia coli Cu,Zn superoxide dismutase reveals the absence of the copper-coordinated water molecule. is the water molecule really necessary for the enzymatic mechanism?

The active site of the Cu,Zn superoxide dismutase from Escherichia coli in the oxidized Cu(II) state has been studied by nuclear magnetic relaxation dispersion (NMRD), optical and nuclear magnetic resonance spectroscopy. The orientation of some metal ligands is different with respect to all the other Cu,Zn superoxide dismutases. Moreover, NMRD measurements demonstrate the lack of a copper-coordinated water molecule. In spite of these differences the enzymatic activity is still high. Azide also binds copper with normal affinity and induces modifications in the active site comparable to those previously observed in the eukaryotic enzymes. Our results suggest that, in this enzyme, the copper-coordinated water molecule appears not necessary for the enzymatic reaction. A role for the copper-coordinated water molecule is discussed in the light of recent crystallographic studies.

Binding Sites↗

PubMed2000

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 Parkinson's 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.

Aged↗

PubMed2000

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 Parkinson's 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 Parkinson's 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 Parkinson's disease.

Aged↗

PubMed2000

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 Parkinson's disease patients have been investigated. Results show different relaxometric behavior between the groups, which is consistent with protein aggregation in Parkinson's disease specimens.

Adult↗

PubMed1999

Metal complexes as allosteric effectors of human hemoglobin: an NMR study of the interaction of the gadolinium(III) bis(m-boroxyphenylamide)diethylenetriaminepentaacetic acid complex with human oxygenated and deoxygenated hemoglobin.

The boronic functionalities on the outer surface of the Gd(III) bis(m-boroxyphenylamide)DTPA complex (Gd(III)L) enable it to bind to fructosamine residues of oxygenated glycated human adult hemoglobin. The formation of the macromolecular adduct can be assessed by NMR spectroscopy via observation of the enhancement of the solvent water proton relaxation rate. Unexpectedly, a strong binding interaction was also observed for the oxygenated unglycated human adult hemoglobin, eventually displaying a much higher relaxation enhancement. From relaxation rate measurements it was found that two Gd(III)L complexes interact with one hemoglobin tetramer (KD = 1.0 x 10(-5) M and 4.6 x 10(-4) M, respectively), whereas no interaction has been observed with monomeric hemoproteins. A markedly higher affinity of the Gd(III)L complex has been observed for oxygenated and aquo-met human adult hemoglobin derivatives with respect to the corresponding deoxy derivative. Upon binding, a net change in the quaternary structure of hemoglobin has been assessed by monitoring the changes in the high-resolution 1H-NMR spectrum of the protein as well as in the Soret absorption band. On the basis of these observations and the 11B NMR results obtained with the diamagnetic La(III)L complex, we suggest that the interaction between the lanthanide complex and deoxygenated, oxygenated, and aquo-met derivatives of human adult hemoglobin takes place at the 2, 3-diphosphoglycerate (DPG) binding site, through the formation of N-->B coordinative bonds at His143beta and His2beta residues of different beta-chains. The stronger binding to the oxygenated form is then responsible for a shift of the allosteric equilibrium toward the high-affinity R-state. Accordingly, Gd(III)L affinity for oxygenated human fetal hemoglobin (lacking His143beta) is significantly lower than that observed for the unglycated human adult tetramer.

Adult↗

PubMed1999

A novel 19F-NMR method for the investigation of the antioxidant capacity of biomolecules and biofluids.

A new assay for the measurement of the antioxidant capacity of biomolecules by high resolution 19F-NMR spectroscopy is presented here. This method is based on the use of trifluoroacetanilidic detectors, namely trifluoroacetanilide, N-(4-hydroxyphenyl)-trifluoroacetamide and 2-hydroxy-4-trifluoroacetamidobenzoic acid. Upon hydroxyl radical attack, such fluorinated detectors yield trifluoroacetamide and trifluoroacetic acid that can be quantitatively determined by 19F-NMR spectroscopy. Trifluoroacetamide was found to be a reliable reporter of hydroxyl radical attack on the fluorinated detectors, whereas N-(4-hydroxyphenyl)-trifluoroacetamide was found to be the most sensitive detector amongst the ones considered. Therefore, N-(4-hydroxyphenyl)-trifluoroacetamide has been used in competition experiments to assess the antioxidant capacity of a number of low and high molecular weight antioxidants. The antioxidant capacity of a given compound has been scaled in terms of an adimensional parameter, kF, that represents the ratio between the scavenger abilities of the fluorinated detector and the competitor. kF values obtained for low-molecular-mass compounds fall in the range 0.17 < kF < 1.5 and are in good agreement with second order rate constants (k2OH) for the reaction of the antioxidant with hydroxyl radicals. The kF value for serum albumin is much larger (46.9) than that predicted from the reported k2OH value. This finding supports the view that the protein can very effectively scavenge hydroxyl radicals as well as secondary radicals. Human blood serum showed that its antioxidant capacity is even higher than that shown by aqueous solutions of albumin at physiologic concentration suggesting a further contribution from other macromolecular serum components.

Antioxidants↗

PubMed1999

Stabilization of the T-state of human hemoglobin by proflavine, an antiseptic drug.

The effect of proflavine (3,6-diaminoacridine), an antiseptic drug, on the spectroscopic and oxygen binding properties of ferrous human adult hemoglobin (Hb) has been investigated. Upon binding of proflavine to the nitric oxide derivative of ferrous human adult hemoglobin (HbNO), the X-band EPR spectrum displays the characteristics which have been attributed to the T-state of the ligated tetramer. In parallel, oxygen affinity for the deoxygenated derivative of ferrous human adult Hb decreases in the presence of proflavine. The effect of proflavine on the spectroscopic and ligand binding properties of ferrous human adult Hb is reminiscent that of 2,3-D-glycerate bisphosphate, the physiological modulator of Hb action.

Allosteric Regulation↗

PubMed1998

Stabilization of the T-state of ferrous human adult and fetal hemoglobin by Ln(III) complexes: a thermodynamic study.

The effect of the lanthanide(III) complexes [Gd(1,4,7,10-tetraazacyclododecane-N,N',N", N"'-tetrakis(methylenephosphonate))]5- (Gd-DOTP) and La-DOTP on the oxygen binding and spectroscopic properties of human adult and fetal hemoglobin (HbA and HbF, respectively) has been investigated. The affinity of Gd-DOTP and La-DOTP for oxygenated HbA (HbAO2; KHbAO2 = 2.6 x 10(-3) M) is closely similar to that observed for Ln(III) complexes association to nitrosylated HbA (HbANO KHbANO = 1.8 x 10(-3) M) and to aquo-met HbA (met-HbA; Kmet-HbA = 1.9 x 10(-3) M), being lower than that determined for Gd-DOTP and La-DOTP binding to the deoxygenated form of the tetramer (HbAd; KHbAd = 3.0 x 10(-4) M). The affinity of Gd-DOTP for deoxygenated HbF (HbFd; KHbFd = 9.5 x 10(-4) M) and oxygenated HbF (HbFO2; KHbFO2 = 3.7 x 10(-3) M) is lower than that observed for Ln(III) complexes association to HbAd and HbAO2, respectively. Gd-DOTP and La-DOTP bind to HbA and HbF with a 1:1 stoichiometry per tetramer. Increasing Gd-DOTP and La-DOTP concentration, oxygen affinity for HbA decreases (i.e. P50 increases), this effect being minor for HbF. Upon binding of Ln(III) complexes to HbANO, the X-band EPR spectrum and the absorption spectrum in the Soret region display the characteristics which have been attributed to the T-state of the ligated tetramer. These results represent a clear cut evidence for the specific binding of Gd-DOTP and La-DOTP to the 2,3-D-glycerate bisphosphate (BPG) pocket (i.e. at the dyad axis, in between the beta-chains) of HbA and HbF. The effect of Ln(III) complexes on the ligand binding and spectroscopic properties of HbA and HbF is reminiscent that of BPG, the physiological modulator of human Hb action.

Adult↗

PubMed1998

MIB-1 as a predictor of response in lung allografts with moderate acute cellular rejection.

The major obstacle that long-term lung transplant recipients face is bronchiolitis obliterans. Prior episodes of acute rejection, specifically their frequency, persistence, and severity, are important predictors of bronchiolitis obliterans. Many cells contribute to the damage of acute rejection, and there is no sole cell type that can predict persistent rejection or bronchiolitis obliterans. In this study we evaluated 48 transbronchial biopsy samples from various grades of acute rejection with the proliferation marker MIB-1 and attempted to retrospectively predict response to standard corticosteroid in a subpopulation of nine responders and nine nonresponders, all with grade A3 rejection. We then characterized the proliferating cells by double labeling with MIB-1 and L26, CD3, OPD4, or KP1. Our results indicate that the proliferating cells in acute lung rejection are a heterogeneous pool of T- and B-lymphocytes, T-helper cells, macrophages, endothelial cells, and possibly parenchymal cells, and that MIB-1 is a valuable tool in the evaluation of total cellular activity in this setting. In addition, the overall proliferation rate, defined as the most intense proliferation rate regardless of location in the biopsy, closely matches the grade of acute rejection. Finally, a low lesional proliferation rate, defined as the proliferation rate at the site of perivascular inflammation diagnostic of acute rejection, is an indicator of excellent response to therapy and may have potential clinical importance.

Adrenal Cortex Hormones↗

PubMed1998

Immunohistochemical evaluation of hepatoblastomas with use of the hepatocyte-specific marker, hepatocyte paraffin 1, and the polyclonal anti-carcinoembryonic antigen.

The distinction of hepatoblastoma, especially the embryonal type, from other small, round-cell tumors of childhood can sometimes be difficult. Polyclonal anticarcinoembryonic antigen (pCEA) and Hepatocyte Paraffin 1 (Hep Par 1) are immunohistochemical markers that are useful in the diagnosis of hepatocellular carcinomas. We immunohistochemically studied pCEA, monoclonal CEA (mCEA), and Hep Par 1 on 12 hepatoblastomas (3 fetal type, 2 embryonal type, and 7 mixed epithelial type). In addition, we studied the expression of Hep Par 1 on 27 other selected childhood tumors, including 1 hepatocellular carcinoma, 5 germ-cell tumors, 4 peripheral neuroectodermal tumors/Ewing's sarcomas, 3 rhabdomyosarcomas, 5 neuroblastomas, 2 rhabdoid tumors, 3 lymphomas, and 4 Wilms' tumors. All of the hepatoblastomas expressed Hep Par 1 with a characteristic granular intracytoplasmic pattern that was generally less intense in embryonal-type than in fetal-type hepatoblastomas, perhaps reflecting the degree of hepatocyte differentiation. All of the fetal-type hepatoblastomas expressed pCEA with both an intracytoplasmic and bile canalicular pattern. Embryonal type hepatoblastomas were more likely to be pCEA negative or to show focal or no canalicular pattern of expression, again possibly reflecting the degree of hepatocyte differentiation. All of the hepatoblastomas were mCEA negative. All of the nonhepatoblastomas were Hep Par 1 negative, except for the one hepatocellular carcinoma in this study, which was Hep Par 1 positive. We conclude that Hep Par 1 and pCEA are useful markers for hepatoblastomas, as they have been shown to be in hepatocellular carcinomas.

Adolescent↗

PubMed1997

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.

Brain Chemistry↗

PubMed1997

CD44 and its v6 spliced variant in lung tumors: a role in histogenesis?

BACKGROUND: CD44 is a polymorphic family of cell surface glycoproteins with a variety of functions including participation in cell adhesion and migration as well as modulation of cell-matrix interactions. Expression of the standard form of CD44 (CD44s) and its variant isoforms has been shown in both normal and neoplastic tissue and holds promise as a prognostic indicator. METHODS: The authors investigated the expression of CD44s and its v6 isoform (CD44v6) immunohistochemically in 7 fetal lungs (gestational age between 11-36 weeks) and in 80 lung tumors of various histologic types, degrees of differentiation, and clinical stages. RESULTS: In the fetal lung, CD44v6 was expressed as membranous and luminal staining of epithelial cells throughout gestation and basal staining of bronchial epithelium late in gestation. Nonneoplastic adult lung showed CD44v6 expression that was restricted to epithelial cells with membranous staining of basal bronchial cells and squamous metaplasia as well as basolateral membranous staining of type 2 pneumocytes. CD44s showed similar but less intense staining and was in addition present on lymphocytes and macrophages. Tumorlets and neuroepithelial bodies were CD44v6 negative. Nearly all squamous cell carcinomas (97%) were positive for CD44v6 with patterns similar to squamous metaplasia and with more intense staining at the periphery of tumor nests. Most adenocarcinomas (90%) were CD44v6 negative whereas most bronchioloalveolar cell carcinomas (71%) were CD44v6 positive with patterns similar to that in type 2 pneumocytes. Most large cell carcinomas (71%), carcinoid tumors (67%), and all small cell carcinomas were CD44v6 negative. CD44v6 expression did not correlate with clinical stage. CD44v6 expression in lymph node metastases was identical to that of the primary tumor. CONCLUSIONS: The results of the current study show that CD44v6 is localized differently in fetal and adult lung, suggesting a difference in function. In the fetal lung, it may modulate growth factors important in morphogenesis and maturation. In the adult nonneoplastic lung, CD44v6 is associated with stem cells, namely basal cells and type 2 pneumocytes, and may act to anchor these cells to the matrix and be important in migration during repair or neoplasia. In addition, CD44v6 expression is maintained throughout tumorigenesis in squamous cell carcinoma and bronchioloalveolar cell carcinoma, suggesting a histogenetic relationship between the stem cells and the respective tumors. Conversely, most neuroendocrine tumors and the cells of the dispersive neuroendocrine system do not express CD44v6, implying a separate histogenetic lineage in these tumors.

Adult↗