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Biomedical subjects

F Neri

Publications and source records attributed to F Neri.

At least 19 recordsLinked to original sources
PubMed2000

Effect of pH on axial ligand coordination of cytochrome c" from Methylophilus methylotrophus and horse heart cytochrome c.

The effect of protons on the axial ligand coordination and on structural aspects of the protein moiety of cytochrome c' ' from Methylophilus methylotrophus, an obligate methylotroph, has been investigated down to very low pH (i.e., 0.3). The unusual resistance of this cytochrome to very low pH values has been exploited to carry out this study in comparison with horse heart cytochrome c. The experiments were undertaken at a constant phosphate concentration to minimize the variation of ionic strength with pH. The pH-linked effects have been monitored at 23 degrees C in the oxidized forms of both cytochromes by following the variations in the electronic absorption, circular dichroism and resonance Raman spectra. This approach has enabled the conformational changes of the heme surroundings to be monitored and compared with the concomitant overall structural rearrangements of the molecule. The results indicate that horse heart cytochrome c undergoes a first conformational change at around pH 2.0. This event is possibly related to the cleavage of the Fe-Met80 bond and a likely coordination of a H(2)O molecule as a sixth axial ligand. Conversely, in cytochrome c" from M. methylotrophus, a variation of the axial ligand coordination occurs at a pH that is about 1 unit lower. Further, it appears that a concerted cleavage of both His ligands takes place, suggesting indeed that the different axial ligands present in horse heart cytochrome c (Met/His) and in cytochrome c" from M. methylotrophus (His/His) affect the heme conformational changes.

Animals↗
PubMed2000

[Retinopathy of prematurity: incidence and risk factors].

The study analyses the incidence of the retinopathy of prematurity (ROP) and its correlation with the most important risk factors in neonates with gestational age (GE) < or = 32 weeks and/or with birth weight (BW) < or = 1500 g. The cohort of our study is composed by 305 preterms with Mean GE of 29.8 weeks and Mean BW of 1312 g, studied at the Neonatal Intensive Care Unit of the University of Parma during January 1993-December 1999. The incidence of ROP resulted 19.7% in our group and was inversely proportional to the GE and BW. No preterms with GE < or = 32 weeks presented retinopathy, while there has been an incidence of the 65% in the subjects with GE lower than 26 weeks. The most severe grade of ROP was mainly seen in the newborns with the lowest GE (31%), whilst only the 1.5% of the preterms with GE of 30-31 weeks developed ROP of stage 3 or 3+. Similar results were seen when the data were compared with the birth weight. However a direct relation was seen with the duration of the O2 therapy and with the typical pathologies of the prematurity, namely respiratory distress syndrome (RDS) and intraventricular haemorrhages (IVH). Among the other risk factors evaluated, the blood transfusion was the most relevant.

Humans↗
PubMed1999

Role of the distal phenylalanine 54 on the structure, stability, and ligand binding of Coprinus cinereus peroxidase.

Resonance Raman and electronic absorption spectra obtained at various pH values for the Fe3+ form of distal F54 mutants of Coprinus cinereus peroxidase are reported, together with the Fe2+ form and fluoride and imidazole adducts at pH 6.0, 5.0, and 10.5, respectively. The distal phenylalanine residue has been replaced by the small aliphatic residues glycine and valine and the hydrogen-bonding aromatic residues tyrosine and tryptophan (F54G, -V, -Y, and -W, respectively). These mutations resulted in transitions between ferric high-spin five-coordinate and six-coordinate forms, and caused a decrease of the pKa of the alkaline transition together with a higher tendency for unfolding. The mutations also alter the ability of the proteins to bind fluoride in such a way that those that are six-coordinate at pH 5.0 bind more strongly than both wild-type CIP and F54Y which are five-coordinate at this pH value. The data provide evidence that the architecture of the distal pocket of CIP is altered by the mutations. Direct evidence is provided that the distal phenylalanine plays an important role in controlling the conjugation between the vinyl double bonds and the porphyrin macrocycle, as indicated by the reorientation of the vinyl groups upon mutation of phenylalanine with the small aliphatic side chains of glycine and valine residues. Furthermore, it appears that the presence of the hydrogen-bonding tyrosine or tryptophan in the cavity increases the pKa of the distal histidine for protonation compared with that of wild-type CIP.

Binding Sites↗
PubMed1998

Spectroscopic characterization of recombinant pea cytosolic ascorbate peroxidase: similarities and differences with cytochrome c peroxidase.

Recombinant pea cytosolic ascorbate peroxidase (APX) has been characterized by resonance Raman (RR) and electronic absorption spectroscopies. The ferric and ferrous forms together with the complexes with fluoride and imidazole have been studied and compared with the corresponding spectra of cytochrome c peroxidase (CCP). Ferric APX at neutral pH is a mixture of 6- and 5-coordinate high-spin and 6-c low-spin hemes, the latter two species being dominant. The results suggest that the low-spin form derives from a water/hydroxo ligand bound to the heme iron and not from a strong internal ligand as observed in CCP at alkaline pH. Two Fe-Im stretching modes are identified, as in CCP, but the RR frequencies confirm a weaker His163-Asp208 hydrogen bond than in CCP, as suggested on the basis of the X-ray structure [Patterson, W. R., and Poulos, T. L. (1995) Biochemistry 34, 4331-4341]. The data show that CCP and APX have markedly different orientations of the vinyl substituents on the heme chromophore resulting from different steric constraints exerted by the protein matrix.

Ascorbate Peroxidases↗
PubMed1998

Mutation of the distal arginine in Coprinus cinereus peroxidase--structural implications.

Heme peroxidases of prokaryotic, plant and fungal origin share the essential His and Arg catalytic residues of the distal cavity and a proximal His bound to heme iron. Spectroscopic techniques, in contrast to X-ray crystallography, are well suited to detect the precise structure, spin and coordination states of the heme as influenced by its near environment. Resonance Raman and electronic absorption spectra obtained at various pH values for Fe3+ and Fe2+ forms of distal Arg51 mutants of the fungal Coprinus cinereus peroxidase are reported, together with the fluoride adducts at pH 5.0. This basic catalytic residue has been replaced by the aliphatic residue Leu, the polar residues Asn and Gln and the basic residue Lys (Arg51-->Leu, Asn, Gln, and Lys, respectively). These mutations cause changes in the coordination and spin states of the heme iron, and in the v(Fe-Im) stretching frequency. The variations are explained in terms of pH-dependent changes, charge location, size and hydrogen-bonding acceptor/donor properties of the residue at position 51. The present work indicates that the hydrogen-bond capability of the residue in position 51 influences the occupancy of water molecules in the distal cavity and the ability to form stable complexes between anionic ligands and the heme Fe atom.

Amino Acid Sequence↗
PubMed1997

pH dependence of structural and functional properties of oxidized cytochrome c" from Methylophilus methylotrophus.

Cytochrome c" from Methylophilus methylotrophus is an unusual monoheme protein that undergoes a major redox-linked change in the heme arrangement: one of the two axial histidines bound to the iron in the oxidized form is detached upon reduction and a proton is taken up. The kinetics of reduction by sodium dithionite and the spectroscopic properties of the oxidized cytochrome c" have been investigated over the pH range between 1.4 and 10.0. The rate of reduction displays proton-linked transitions of pKa congruent with 5.5 and 2.4, and a spectroscopic transition with a pKa congruent with 2.4 is also observed. The protein displays a complete reversibility after exposure to low pH, and both electronic absorption and resonance Raman spectroscopic properties suggest that the transition at lower pH brings about a drastic change in the heme coordination geometry. Circular dichroism spectra indicate that over the same proton-linked transition, the protein undergoes a marked decrease (approximately 60%) of the alpha-helical content toward a random coil arrangement, which is recovered upon increasing the ionic strength. The structural change at low pH is linked to a concerted two-proton transition, suggesting the detachment and protonation of axial histidine(s). Such kinetic and spectroscopic features along with the remarkable capacity of this protein to recover its native structure after exposure to extremely low pH values makes it a promising model for studying folding processes and stability in heme proteins.

Circular Dichroism↗
PubMed1997

Fluoride binding in hemoproteins: the importance of the distal cavity structure.

The electronic absorption and resonance Raman spectra of the fluoride complexes of various peroxidases and selected site-directed mutants have been studied at pH 5.0, and compared to the spectra obtained for the myoglobin-F adduct. It is shown that the electronic absorption maxima depend on the degree of conjugation between the porphyrin macrocycle and the vinyl substituents. Moreover, it is confirmed that the wavelength of the CT1 band is a sensitive probe of axial ligand polarity and of its interaction with the distal protein residues. The results highlight the different mechanism of stabilization of the fluoride ligand exerted by the distal residues in myoglobin and peroxidases. In peroxidases, the Arg is determinant in controlling the ligand binding via a strong hydrogen bond between the positively charged guanidinium group and the anion. Mutation of Arg to Leu decreases the stability of the complex by 900-fold, suggesting that this interaction stabilizes the complex by 4 kcal/mol. The distal His also contributes to the stability of the fluoride complex, presumably by accepting a proton from HF and hydrogen-bonding, through a water molecule, to the anion. Mutation of His to Leu decreases the stability of the fluoride complex by 30-fold, suggesting that this interaction is much weaker than the interaction with the distal Arg. For Mb, the distal His is solely responsible for stabilization of the exogenous ligand.

Binding Sites↗
PubMed1997

The study of visual evoked potentials in patients with thyroid-associated ophthalmopathy identifies asymptomatic optic nerve involvement.

In the present study we have recorded visual evoked cortical potentials (VECP) in 88 patients affected by autoimmune thyroid disease and thyroid-associated ophthalmopathy (TAO) without clinical signs of optic neuropathy. At the time of ophthalmological examination, 37 of these patients were hyperthyroid, 41 were euthyroid, and 8 were hypothyroid; 2 were not assessed. Twenty-nine normal subjects served as controls. We performed pattern reversal visual stimulation and recorded the amplitude and latency of the cortical electric response at 100 ms (P100 wave). There were no differences in the mean P100 amplitude of TAO patients and normal subjects. The mean P100 latency in patients was 105.6 +/- 0.5 ms, significantly higher than that in normal subjects (102.0 +/- 0.5 ms; P < 0.00003). Latency in euthyroid patients did not differ from that in either hypo- or hyperthyroid patients. The VECP test was positive (latency, > or = 110.0 ms) in 21 (23.8%) TAO patients. In patients with proptosis greater than 21 mm, latency was 106.7 +/- 0.7 ms, significantly higher than that in patients with normal Hertel measurements (104.3 +/- 0.6 ms; P < 0.01). Latency was not increased in patients with acute inflammatory signs compared to those with inactive eye disease and in patients with altered extrinsic motility. In patients with an abnormal visual field study, the mean latency was 110.3 +/- 1.5 ms, significantly higher than that in patients with a normal visual field (104.7 +/- 0.4; by t test, P < 0.000003). In conclusion, we observed a prolongation of the latency of the evoked cortical response in patients with TAO without subjective visual complaints and without optic nerve compression. We believe that the study of VECP in TAO is complementary to the study of the visual field in identifying early optic nerve dysfunction in the absence of decreased visual acuity.

Adolescent↗
PubMed1996

Versatility of heme coordination demonstrated in a fungal peroxidase. Absorption and resonance Raman studies of Coprinus cinereus peroxidase and the Asp245-->Asn mutant at various pH values.

The pH dependence of the electronic absorption and resonance Raman (RR) spectra of FeIII and FeII forms of Coprinus cinereus peroxidase (CIP) and its Asp245-->Asn (D245N) mutant has been examined in detail. The spectral data were obtained in the pH range 3.8-12.0. These spectra were used to assess the spin and ligation states of the heme via the porphyrin marker band frequencies and the wavelengths of the absorption maxima, especially that of the band (CT1) due to the charge transfer from the porphyrin to the heme iron via the a' 2u(pi)-->eg (d pi) electronic transition. The RR spectra were obtained by using different excitation wavelengths and polarized light. The data obtained for ferric CIP show that two pH-induced structural transitions exist. At acid pH the Soret and the CT1 absorption maxima occur at 394 and 652 nm, respectively, compared with the values of 403 and 649 nm observed at neutral pH. The electronic data indicate that at acid pH the proximal Fe-Im bond might be weakened or ruptured, and the RR spectra show a new species (5-c HS) different from the normal neutral 5-coordinate high-spin (5-c HS) heme. At pH 12.0, the protein converts to a 6-coordinate low-spin (6-c LS) heme with a hydroxyl ligand coordinated in the sixth position of the heme iron and strongly hydrogen-bonded with the positively charged guanidinium group of the distal Arg51 residue. Replacement of the aspartate carboxylate group of Asp245, which acts as hydrogen-bond acceptor to the proximal His183 ligand of the heme Fe, with a carboxamide group of an asparagine residue has a profound influence on the heme coordination. The RR spectra of the Fe(II) form of this mutant at both neutral and alkaline pH values show a band at 204 cm-1 assigned to the Fe-His stretch associated with a fairly weak or non-hydrogen-bonded imidazole. The ferric form of the mutant shows a great variability in coordination and spin states upon pH titration. Between pH 8.8 and 3.8 the spectra are mainly characteristic of a 6-coordinate high-spin heme, presumably with a water molecule bound on the distal side of the Fe atom. The pKa of the alkaline transition of the mutant is much lower than that of the wild-type protein. At pH 10.0 the D245N mutant is in its final alkaline form, which markedly differs from that of the parent enzyme. The spectral data indicate that the majority of the protein has 5-coordinate high-spin heme (5-c HS), with the Fe-His 183 bond broken and the distal axial coordination site of the heme iron occupied by a hydroxyl group, which is strongly hydrogen-bonded with distal Arg51. Therefore, the Asp245-->Asn mutation on the proximal side results in the breakage of the Fe-His bond at alkaline pH.

Asparagine↗
PubMed1995

Effect of the His175-->Glu mutation on the heme pocket architecture of cytochrome c peroxidase.

Resonance Raman (RR) and electronic absorption spectra of the ferric and ferrous forms of the His175Glu mutant of cytochrome c peroxidase are reported. At 296 K, the FeIII form is five-coordinate high spin and the resonance Reman spectra are very similar to those obtained for the wild type enzyme, even though in the mutant the Fe atom is bound to an oxygen atom of the Glu residue. The only difference is that the bands due to the out-of-plane modes are very weak, indicating a less distorted heme plane compared to CCP. The absorption spectrum is similar to that of CCP, as far as the Soret and alpha, beta bands are concerned, but the charge-transfer band due to the a2u(pi)-->eg(d pi) transition is 8 nm blue-shifted relative to that of the wild type enzyme, indicating that a more negative ligand is bound to the heme iron. As the temperature is lowered, the five-coordinate heme converts to a six-coordinate high-spin form. The conversion is readily reversible. A temperature effect on the protein structure is proposed that permits the Fe atom to approach the heme plane and to bind the distal water molecule. The results are discussed in terms of the X-ray structure, which shows a different disposition of the distal water molecules in the Glu175 mutant. The RR spectra also show that the heme is more contracted and distorted at 19 K than at room temperature.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗
PubMed1994

Echographic diagnosis of pretibial myxedema in patients with autoimmune thyroid disease.

In the present study we have evaluated the use of pretibial ultrasound for the diagnosis of pretibial myxedema (PTM). We studied 76 patients, 58 with Graves' disease, 13 with Hashimoto's thyroiditis and five with idiopathic hypothyroidism. Thirty-two normal subjects were also studied as controls. Sixty-four patients had associated ophthalmopathy. The ultrasound scanner was equipped with 10- and 13-MHz probes. Punch biopsies were carried out in 11 patients and tissue sections examined on a light microscope. On clinical examination 21 patients (28%) had suspected PTM. By ultrasound, we measured the thickness of dermis and subcutaneous tissue (D1) and that including only deeper dermis (D2) in normal subjects to define the echographic parameters of normal pretibial skin. We then found increased skin thickness in 25 patients (33%), with mean D1 and D2 values significantly higher than those measured in controls (p < 0.00001). The echographic study was positive in 20 patients with ophthalmopathy (31%). Ultrasound showed increased skin thickness in 16 of 21 patients (76%) with clinically suspected PTM. Histopathological findings confirmed the presence of PTM in all the patients who underwent pretibial skin biopsy. We believe that the measurement of pretibial skin thickness by ultrasound may be useful for revealing the presence of PTM.

Adult↗