Biomedical subjects
Publications and source records attributed to G Gilardi.
Three Fusarium wilts recently observed in Italy on lettuce (Lactuca sativa), wild (Diplo taxis spp.) and cultivated (Eruca sativa) rocket and lamb's lettuce (Valerianella olitoria) emerged as major production problems in Lumbardy (north-western Italy). Aspects of biology and epidemiology of the three diseases and some possibilities of disease management are discussed.
Redox proteins and enzymes are attractive targets for nanobiotechnology. The theoretical framework of biological electron transfer is increasingly well-understood, and several properties make redox centres good systems for exploitation: many can be detected both electrochemically and optically; they can perform specific reactions; they are capable of self-assembly; and their dimensions are in the nanoscale. Great progress has been made with the two main approaches of protein engineering: rational design and combinatorial synthesis. Rational design has put our understanding of the structure-function relationship to the test, whereas combinatorial synthesis has generated new molecules of interest. This article provides selected examples of novel approaches where redox proteins are "wired up" in efficient electron-transfer chains, are "assembled" in artificial multidomain structures (molecular Lego), are "linked" to surfaces in nanodevices for biosensing and nanobiotechnological applications.
Engineered metalloproteins offer interesting systems for electrochemical studies of protein structure/function and their applications in nanobiotechnology. Scanning probe microscopy and cyclic voltammetry of engineered metalloproteins and electrodes have proved to be a powerful combination of tools contributing to the field of bioelectrochemistry. The ability to engineer tags, such as histidine tags and biotin-acceptor peptides, and to site-specifically introduce cysteine residues enabled the creation of ordered immobilised protein structures that can be characterised both electrochemically and topographically. Gene fusion and de novo combinatorial synthesis of metalloproteins are emerging to provide structures with the desired electrochemical properties.
The recent identification of a common etiology among MASA syndrome (McKusick 303300), X-linked hydrocephalus (HSAS) (McKusick 307000) and other related neurological disorders, which had previously been considered distinct nosological entities, allowed us to diagnose MASA syndrome in a male fetus in a primigravida at the 29th week of gestation by sonographic signs of the MASA spectrum such as hydrocephalus and hypoplasia of corpus callosum. Indeed, the evidence of an X-linked neurological disease in the brother and the maternal uncle of the pregnant women enabled us to estimate a 25% risk of a male fetus being an affected hemizygote. The way in which a prenatal diagnosis, based on instrumental procedures, was reached is described since the authors were unable to perform, at the time of the observation, a molecular confirmation which was carried out only after birth.
A hypothetical model for the non-physiological electron transfer complex between cytochrome c553 (c553) and the flavodoxin (fld) from the sulphate-reducing bacteria Desulfovibrio vulgaris has been recently published [1] based on rigid-body docking and refined by molecular dynamics. In this study, the functional validity of this model is tested by looking at the role of electrostatics in the non-physiological interprotein electron transfer between the two proteins at different ionic strengths. The results are compared with the electron transfer between fld and cytochrome c from horse heart (hhc). Second-order rate constants (k2) were measured for both non-physiological systems at different ionic strengths: a complex, bell-shaped behaviour is observed for the k2 of the c553/fld redox pair with an optimum rate at I=58 mmol l(-1), whereas under the same conditions the k2 for hhc/fld decreased monotonically with increasing ionic strength. Results from the electron transfer kinetics are rationalised in terms of reorganisational effects of an ensemble of conformations of the electron transfer competent c553/fld complexes, consistent with the published model.
This work reports on a novel approach for building artificial redox chains: the molecular 'Lego' approach. This exploits the scaffold of natural redox proteins by fusing together functional protein modules with the desired properties. The molecular 'Lego' mimics the natural molecular evolution that proceeded by modular assembly of genes/DNA segments. Non-physiological electron transfer partners, flavodoxin (fld) and cytochrome c553 (c553) from Desulfovibrio vulgaris and the haem domain of P450 BM3 (BMP) from Bacillus megaterium have been used as building blocks in different combinations to build artificial redox chains. The kinetic characterization of the electron transfer (ET) between the separate building blocks has been carried out. Under pseudo-first order conditions, a limiting ET rate, klim, of 0.48 +/- 0.05 s-1 and 43.77 +/- 2.18 s-1 and an apparent binding constant, Kapp, of 21 +/- 6 microM and 1.23 +/- 0.32 microM have been found for the fld/c553 and fld/BMP redox pairs, respectively. These results show that fld can be used as a module for transferring electrons to c553 and BMP. A 3D model of the fld/c553 and fld/BMP complexes was used to guide the construction of covalently linked assemblies via engineered disulfide bridges or by fusion of the relevant genes via an engineered loop. The first approach led to the construction, expression and characterization of the S35C and S64C mutants of fld and M23C and G51C mutants of c553. Although the redox potentials of the separate mutants were found to be the same as those of recombinant wild type proteins (-408 mV for the semiquinone/hydroquinone couple of fld and +32 mV for the c553), the c553 homo-dimers M23C-M23C and G51C-G51C were found to have redox potentials of +88 and +105 mV, respectively. These differences have been analysed in terms of exposure of the haem cofactors to the solvent, and these lead to some interesting questions on the redox potentials of the transient redox complexes in physiological systems. The fld-c553 S64C-M23C and S35C-M23C chimeras were constructed, expressed and purified but the FMN was found to be destabilised resulting in the apo-form of these proteins. The gene fusion strategy was used to produce covalently linked assemblies of both fld-c553 and fld-BMP. The former was expressed using a seven amino acid (GPGPGPG) loop linking the C-terminus of fld to the N-terminus of c553. The fld-BMP fusion protein was successfully expressed by using the naturally occurring loop of the P450 BM3 (residues 471-479) to link the BMP domain at the N-terminus with fld domain at the C-terminus. This fusion was found to be correctly folded and functional. Efficient ET from the FMN to the haem domain (370 s-1) was also found to be in the same region of the physiological redox partners (250 s-1). This work demonstrates the feasibility of the molecular 'Lego' approach in generating functional multi-domain proteins with designed properties, beyond the restrictions imposed by the naturally occurring protein domains.
15N relaxation measurements have been performed on the type Iota blue copper protein azurin from Pseudomonas aeruginosa. The relaxation times show that one loop (residues 103-108) and one turn (residues 74-77) display fast internal motions. The rest of the protein is rigid with an average order parameter S(2) of 0.85 +/- 0. 05. The copper binding site shows the same degree of rigidity even though is it composed of several loops and lies outside the beta-sheet sandwich. Substantial exchange broadening was found for a number of residues surrounding the side chain of His-35. The average exchange rate has been determined from NMR exchange spectroscopy experiments and is 45 +/- 6 s(-)(1) at 41 degrees C. The exchange broadening is caused by the protonation/deprotonation equilibrium of His-35. The NMR results indicate that the two structures of azurin observed by X-ray diffraction of crystals at pH 5.5 and 9.0 [Nar, H., Messerschmidt, A., Huber, R., Van de Kamp, M., Canters, G. W. (1991) J. Mol. Biol. 221, 765-772] are present in solution and that they interconvert slowly.
Theoretical studies of protein-protein association and electron transfer were performed on the binary systems formed by Desulfovibrio vulgaris Hildenborough (D. v. H.) flavodoxin and D. v. H. cytochrome c553 and by flavodoxin and horse heart cytochrome c. Initial structures for the complexes were obtained by rigid-body docking and were refined by MD to allow for molecular flexibility. The structures thus obtained were analysed in terms of their relative stability through the calculation of excess energies. Electrostatic, van der Waals and solvation energy terms showed all to have significant contributions to the stability of complexes. In the best association solutions found for both cytochromes, these bind to different zones of flavodoxin. The binding site of flavodoxin observed for cytochrome c is in accordance with earlier works [27]. The various association modes found were characterised in terms of electron transfer using the Pathways model. For complexes between flavodoxin and horse heart cytochrome c, some correlation was observed between electron tunnelling coupling factors and conformation energy; the best conformation found for electron transfer corresponded also to the best one in terms of energy. For complexes between flavodoxin and cytochrome c553 this was not the case and a lower correlation was observed between electron tunnelling coupling factors and excess energies. These results are in accordance with the differences in the experimental dependence of electron transfer rates with ionic strength observed between these two cases.
This paper reports the isolation and characterization of phenol hydroxylase (PH) from a strain belonging to the Acinetobacter genus. An Acinetobacter radioresistens culture, grown on phenol as the only carbon and energy source, produced a multicomponent enzyme system, located in the cytoplasm and inducible by the substrate, that is responsible for phenol conversion into catechol. Because of the wide diffusion of phenol as a contaminant, the present work represents an initial step towards the biotechnological treatment of waste waters containing phenol. The reductase component of this PH system has been purified and isolated in large amounts as a single electrophoretic band. The protein contains a flavin cofactor (FAD) and an iron-sulfur cluster of the type [2Fe-2S]. The function of this reductase is to transfer reducing equivalents from NAD(P)H to the oxygenase component. In vitro, the electron acceptors can be cytochrome c as well as other molecules such as 2, 6-dichlorophenolindophenol, potassium ferricyanide, and Nitro Blue tetrazolium. The molecular mass of the reductase was determined to be 41 kDa by SDS/PAGE and 38.8 kDa by gel permeation; its isoelectric point is 5.8. The N-terminal sequence is similar to those of the reductases from A. calcoaceticus NCIB 8250 (10/12 identity) and Pseudomonas CF600 (8/12 identity) PHs, but much less similar (2/12 identity) to that of benzoate dioxygenase reductase from A. calcoaceticus BD413. Similarly, the internal peptide sequence of the A. radioresistens PH reductase displays a good level of identity (9/10) with both A. calcoaceticus NCIB 8250 and Pseudomonas CF600 PH reductase internal peptide sequences but a poorer similarity (3/10) to the internal peptide sequence of benzoate dioxygenase reductase from A. calcoaceticus BD413.
The unfolding of the blue-copper protein azurin from Pseudomonas aeruginosa by guanidine hydrochloride, under nonreducing conditions, has been studied by fluorescence techniques and circular dichroism. The denaturation transition may be fitted by a simple two-state model. The total free energy change from the native to the unfolded state was 9.4 +/- 0.4 kcal.mol-1, while a lower value (6.4 +/- 0.4 kcal.mol-1) was obtained for the metal depleted enzyme (apo-azurin) suggesting that the copper atom plays an important stabilization role. Azurin and apo-azurin were practically unaffected by hydrostatic pressure up to 3000 bar. Site-directed mutagenesis has been used to destabilize the hydrophobic core of azurin. In particular either hydrophobic residue Ile7 or Phe110 has been substituted with a serine. The free energy change of unfolding by guanidinium hydrochloride, resulted to be 5.8 +/- 0.3 kcal.mol-1 and 4.8 +/- 0.3 kcal.mol-1 for Ile7Ser and Phe110Ser, respectively, showing that both mutants are much less stable than the wild-type protein. The mutated apoproteins could be reversible denatured even by high pressure, as demonstrated by steady-state fluorescence measurements. The change in volume associated to the pressure-induced unfolding was estimated to be -24 mL.mol-1 for Ile7Ser and -55 mL.mol-1 for Phe110Ser. These results show that the tight packing of the hydrophobic residues that characterize the inner structure of azurin is fundamental for the protein stability. This suggests that the proper assembly of the hydrophobic core is one of the earliest and most crucial event in the folding process, bearing important implication for de novo design of proteins.
Ascorbate oxidase is a copper-containing enzyme which catalyzes a redox reaction between vitamin C and molecular oxygen. The protein, which shows a complex tertiary structure, is an homodimer of monomers, each containing three domains and 14 tryptophan residues. Recently, we have demonstrated by spectroscopic and ultracentrifugation techniques the existence of a stable dimeric intermediate along the unfolding pathway of this enzyme [Mei, G., Di Venere, A., Buganza, M., Vecchini, P., Rosato, N. & Finazzi Agrò, A. (1997) Biochemistry 36, 10917-10922]. In this study, the steady-state and dynamic fluorescence features of ascorbate oxidase have been exploited in order to find a way of monitoring the individual subsystems of the protein. The fluorescence intensity and anisotropy upon excitation at 295 nm are extremely sensitive functions of the emission wavelength, indicating a great heterogeneity of the system. The emission decay collected through a cut-off filter can be analyzed in terms of two continuous distributions of lifetimes. Using a monochromator in emission or an optical multichannel analyzer, the two distributions may be attributed to distinct components of the fluorescence spectrum. Differential quenching by cesium chloride also confirmed that the several tryptophan residues present in the protein structure may be grouped into two main classes, each with a different environment. Once the complex fluorescence decay of ascorbate oxidase was analyzed and resolved, a comparison with the crystallographic data allowed a first, approximate attribution of the protein spectroscopic properties to some of the tryptophan residues. This might provide a powerful tool of investigation about the role of definite segments of the protein in its three-dimensional structure and catalytic activity. Furthermore, the methodology set up for ascorbate oxidase can be usefully extended to other multitryptophan proteins.
This work demonstrates that non-physiological electron transfer (ET) can occur in solution between wild type D. vulgaris flavodoxin (Fld) and horse heart cytochrome c (cyt-c), D. vulgaris cytochrome c553 (cyt-c553) and the haem domain of B. megaterium cytochrome P450 (cyt-P450 BMP). Second order rate constants of the ET reaction between [Fld]sq/[cyt-c]ox, [Fld]sq/[cyt-c553]ox and [Fld]sq/[cyt-P450 BMP]ox, were found to be 6.16 x 10(5), 1.80 x 10(4) and in the region of 10(5) respectively. These data are interpreted in terms of complementarity between the surfaces of the two proteins, their surface and redox potentials. Analysis of the ET results obtained from the separate wild type proteins supported the rational design approach in the creation of Fld-based chimeras. The preliminary design of the chimeras reported here is a 3D prototype for an artificial flavo-cytochrome obtained by covalent linkage of a Fld module to cyt-c553 via a disulphide bond. Theoretical ET rates calculated on the modelled flavo-cytochrome are encouraging the construction of these chimeric systems at DNA level. This work is now underway. The relevance of this molecular lego approach is to be seen in the long term goal of producing engineered multi-domain systems to be applied in the field of biosensors and bioelectronics to fulfil specific requirements. Novel catalytic devices can be obtained by using natural redox proteins in different combinations: this process mimics the natural evolution of proteins such as gene shuffling and gene fusion.
Time-resolved fluorescence and time resolved fluorescence anisotropy studies have been performed on wild-type azurin from Pseudomonas aeruginosa and two variants to study the mobility of Trp48. The two azurin variants in which the microenvironment of Trp48 was changed comprised the single mutations Ile7Ser and Phe110Ser. The experiments were performed on the holo-Cu(I), holo-Cu(II), and apo- forms at various pH values, viscosities, and temperatures; two distinct parts of the emission spectrum were selected for detection. Two prominent subnanosecond lifetimes in the fluorescence decays of the Cu(II) proteins could be observed. The decay of apo-azurin also consists of more than one component. The occurrence of more than one component in the fluorescence decays is explained by conformational heterogeneity. The anisotropy decay results appeared to be different for wild-type and mutated azurins. Phe110Ser and Ile7Ser azurin show more mobility of the Trp48 residue, as reflected in the order parameter.
We describe a case of spontaneous dissecting aneurysm of the circumflex coronary artery in a 44-year-old primigravida at term with no vascular risk factors. Spontaneous coronary artery dissection is a very rare condition with a greater prevalence in women, postpartum in particular. The left anterior descending artery is the most frequently affected. Histologically, the most common finding is a hematoma occupying the outer third of the media, resulting in complete compression of the true lumen. Arterial wall changes during pregnancy together with hemodynamic factors, a lytic action of protease released from eosinophils, and intimal tears are the main hypotheses considered to explain the etiology of spontaneous coronary artery dissection.
Radiosurgical cervical excision has been performed in 59 patients affected by CIN of various stages. This non-traumatic method uses 3.8 MHz radio waves to cut and/or coagulate without postoperative pain and tissue destruction. The radiosurgical excision has been performed in local anesthesia by a loop electrode or microneedle according to the seat of the lesion. The therapy was "effective" in 98% of cases (58/59) whereas the "incomplete excision" of CIN was observed in 6.7% (4/59). Complications (postoperative and late bleeding) occurred in 3.2% of the patients (2/59). Therefore, the Radiosurgical Excision Procedure (REP) can be considered an easy technique in ambulatory surgery; this method is effective in the treatment of CIN with either therapeutic and economic advantages and guarantee the functional and anatomic integrity of the cervix.
The kinetics of a range of ferrocene derivatives with horseradish peroxidase (HRP), cytochrome c peroxidase (CCP) and 3 charge reversal mutants of cytochrome c peroxidase were measured using cyclic voltammetry. Substantial differences in rate constant (100 fold) were observed between HRP and CCP for the same mediator with smaller differences (4-5 fold) for different mediators with the same enzyme. The rate constant did not seem to be dependent on redox potential differences. Cluster analysis is proposed as a way of classifying mediator reactivity.
The maltose binding protein (MBP) has been site specifically labelled with a nitrobenzoxadiazole (NBD) group following mutation of a serine to a cysteine residue at position 337. The resulting protein shows a large ligand (maltose or beta-cyclodextrin) dependent increase in its steady-state fluorescence intensity. Analysis of the static (intensity and anisotropy) and dynamic (lifetime distributions) fluorescence of the NBD label as well as the tryptophan residues in both ligand-bound and ligand-free states of this molecule reveals complex multi-component decays that are interpreted in terms of a ligand-induced solvent shielding mechanism. In the context of the known crystal structures of the various forms of the maltose-binding protein (MBP), ligand-dependent changes in both the fluorescence parameters as well as the circular dichroism spectra of the NBD group are interpreted by a twisted intramolecular charge-transfer (TICT) mechanism, wherein ligand binding locks the NBD group into a conformation that prevents efficient relaxation of the excited state.
A 58-year-old postmenopausal woman with high plasma testosterone levels and virilization, as demonstrated by hirsutism and alopecia, is presented. Urinary 17-ketosteroids and 17-hydroxycorticosteroids as well as the computed axial tomography scan of the adrenal glands were normal. Although no pelvic mass was detected by sonography or pelvic examination, the patient was found to have small pure Leydig cell tumour of the left ovary. Following total abdominal hysterectomy and bilateral salpingo-oophorectomy, the patient had regression of the hirsutism, and the plasma testosterone dropped to normal level.