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Biomedical subjects

B Filippi

Publications and source records attributed to B Filippi.

At least 19 recordsLinked to original sources
PubMed2005

Aluminum-triggered structural modifications and aggregation of beta-amyloids.

We investigated the structural effects induced by Al3+ on different beta-amyloid (Abeta) fragments at pH 7.4 and T=25 degrees C, with particular attention given to the sequences 1-40 and 1-42. Al3+ caused peptide enrichment in beta sheet structure and formation of solvent-exposed hydrophobic clusters. These intermediates evolved to polymeric aggregates which organized in fibrillar forms in the case of the Al3+-Abeta(1-42) complex. Comparative studies showed that Zn2+ and Cu2+ were much less efficient than Al3+ in stimulating the spontaneous aggregation/fibrillogenesis of Abetas. Studies with liposomes as membrane models showed dramatic changes in the structural properties of the lipid bilayer in the presence of Al3+-Abeta complexes, suggesting a major role of Al3+ in Abeta-induced cell dysfunction. Al3+ effects were abolished by desferrioxamine mesylate (DFO) only in solution. We concluded that, in vivo, DFO may act as a protective agent by preventing or reverting Abeta aggregation in the extracellular spaces.

Aluminum↗
PubMed2001

Deflavination of flavo-oxidases by nucleophilic reagents.

Using spectroscopic techniques we studied the effect of the nucleophilic reagents cyanide, cyanate and thiocyanate on three flavo-oxidases namely alcohol oxidase (AO), glucose oxidase (GOX) and D-amino acid oxidase (DAOX). All three ions, added at concentrations in the mM range, caused release of the flavin adenine dinucleotide (FAD) co-factors from the enzyme molecules. In the case of AO this was accompanied by significant conformational perturbations, which was not observed for GOX and DAOX. As suggested from fluorescence, absorption and circular dichroism spectral changes at least one phenolic hydroxyl group became ionized upon FAD release from AO and a new class of Trp residues, fluorescent only in apo-AO protein, was demasked.

Alcohol Oxidoreductases↗
PubMed1999

Conformational transitions accompanying oligomerization of yeast alcohol oxidase, a peroxisomal flavoenzyme.

Alcohol oxidase (AO) is a homo-octameric flavoenzyme which catalyzes methanol oxidation in methylotrophic yeasts. AO protein is synthesized in the cytosol and subsequently sorted to peroxisomes where the active enzyme is formed. To gain further insight in the molecular mechanisms involved in AO activation, we studied spectroscopically native AO from Hansenula polymorpha and Pichia pastoris and three putative assembly intermediates. Fluorescence studies revealed that both Trp and FAD are suitable intramolecular markers of the conformation and oligomeric state of AO. A direct relationship between dissociation of AO octamers and increase in Trp fluorescence quantum yield and average fluorescence lifetime was found. The time-resolved fluorescence of the FAD cofactor showed a rapid decay component which reflects dynamic quenching due to the presence of aromatic amino acids in the FAD-binding pocket. The analysis of FAD fluorescence lifetime profiles showed a remarkable resemblance of pattern for purified AO and AO present in intact yeast cells. Native AO contains a high content of ordered secondary structure which was reduced upon FAD-removal. Dissociation of octamers into monomers resulted in a conversion of beta-sheets into alpha-helices. Our results are explained in relation to a 3D model of AO, which was built based on the crystallographic data of the homologous enzyme glucose oxidase from Aspergillus niger. The implications of our results for the current model of the in vivo AO assembly pathway are discussed.

Alcohol Oxidoreductases↗
PubMed1997

A novel thermostable inhibitor of trypsin and subtilisin from the seeds of Brassica nigra: amino acid sequence, inhibitory and spectroscopic properties and thermostability.

A novel thermostable protein inhibitor of trypsin and subtilisin, called BN, was isolated from the seeds of Brassica nigra. The purified protein gave a single band on SDS-PAGE, corresponding to a molecular mass of 15 500 +/- 1000 Da. The inhibitor is composed of two disulfide-linked polypeptide chains, consisting of 39 and 90 residues, respectively. The amino acid sequence of the two chains was determined by Edman degradation of peptides, isolated from enzyme hydrolysates with TPCK-trypsin, EndoLysC proteinase and a Glu-specific proteinase of reduced and vinylpyridinated protein samples. A segment of the 'heavy' chain, between residues 65 and 81, showed homology with the reactive site loop region of the 6-kDa trypsin inhibitors from Nicotiana alata. The basic residue in position 39 (N. alata) or 70 (napins) is conserved as arginine or lysine in all inhibitors from N. alata and in all napins hitherto sequenced. Probably, the two families of trypsin inhibitors have structurally similar reactive sites. BN exhibits an extremely high thermostability: CD measurements showed that during heating to 97 degrees C it preserves a considerable part of the polypeptide backbone folding. Studies on the fluorescence properties of the inhibitor BN in the absence and presence of neutral or ionic quenchers demonstrated that the intrinsic emission of this protein is dominated by a tryptophyl residue, buried in the interior of the protein matrix. 20% of the light absorbed by Tyr 63 of the 'heavy' chain is transferred to Trp 26 of the 'light' chain.

Amino Acid Sequence↗
PubMed1996

Ineffectiveness of intravenous beta 2-agonists on improving exercise tolerance in patients with reversible chronic airway obstruction.

The effects on exercise tolerance after acute administration of beta 2-agonists were investigated in 11 patients with partly reversible chronic airway obstruction after 400 micrograms of salbutamol (S) given intravenously (i.v.) and after 400 micrograms i.v. of a new selective beta 2-agonist, broxaterol (B), by a cardiopulmonary incremental exercise test. At rest, while VE increased in respect to basal conditions (C) after S (from 13.3 +/- 2.2 to 14.4 +/- 2.8 l/min; p < 0.05) and after B (from 13.6 +/- 3.1 to 15.5 +/- 3.6 l/min; p < 0.05), VO2, VCO2 and VO2/HR showed no substantial variations. A small, not significant reduction of PaO2 was observed both after S (from 82.7 +/- 11.7 to 79.1 +/- 16.7 mm Hg) and B (from 81.6 +/- 10.5 to 78.0 +/- 11.0 mm Hg). The maximum workload increased neither after S (from 67.5 +/- 39.1 to 66.6 +/- 37.0 W) nor after B (from 65.7 +/- 39.3 to 60.0 +/- 35.8 W). At peak of exercise, VO2, VCO2 and VO2/HR did not change after S and B as compared with C, whereas VE remained higher after both beta 2-agonists throughout the effort. VO2 at ventilatory anaerobic threshold (AT) was significantly greater either after S (from 744 +/- 378 to 815 +/- 302 ml/min; p < 0.05) and after B (from 756 +/- 290 to 842 +/- 292 ml/min; p < 0.05). The PaO2 increase shown by these patients during effort was greater after beta 2-agonists administration, delta PaO2 from rest to peak of exercise amounting to 14.9 +/- 14.3 vs. 7.8 +/- 8.2 mm Hg after S and to 17.8 +/- 15.1 vs. 8.8 +/- 10.9 mm Hg after B, in respect to relative baseline (p < 0.05). We conclude that beta 2-agonists, when given acutely, do not improve exercise tolerance in patients with reversible chronic airflow obstruction, although these drugs can induce a small increment of ventilatory AT. In addition, arterial blood gases do not deteriorate at rest and are better preserved during exercise after beta 2-agonists.

Adrenergic beta-Agonists↗
PubMed1995

Synthesis and immunological characterization of 104-mer and 102-mer peptides corresponding to the N- and C-terminal regions of the Plasmodium falciparum CS protein.

We investigated the immunogenicity and the conformational properties of the non-repetitive sequences of the Plasmodium falciparum circumsporozoite (CS) protein. Two polypeptides of 104 and 102 amino acids long, covering, respectively, the N- and C-terminal regions of the CS protein, were synthesized using solid phase Fmoc chemistry. The crude polypeptides were purified by a combination of size exclusion chromatography and RP-HPLC. Sera of mice immunized with the free polypeptides emulsified in incomplete Freund's adjuvant strongly reacted with the synthetic polypeptides as well as with native CS protein as judged by ELISA and IFAT assays. Most importantly, these antisera inhibited the sporozoite invasion of hepatoma cells. In addition, sera derived from donors living in a malaria endemic area recognized the CS 104- and 102-mers. Conformational studies of the CS polypeptides were also performed by circular dichroism spectroscopy showing the presence of a weakly ordered structure that can be increased by addition of trifluoroethanol. The obtained results indicate that the synthetic CS polypeptides and the natural CS protein share some common antigenic determinants and probably have similar conformation. The approach used in this study might be useful for the development of a synthetic malaria vaccine.

Amino Acid Sequence↗
PubMed1995

Stability of subtilisins and related proteinases (subtilases).

The stability towards thermal and chemical (guanidine hydrochloride, GnHCl) denaturation of six inhibited subtilases (mesentericopeptidase, subtilisins BPN', Carlsberg and DY, proteinase K and thermitase) has been investigated by kinetic and equilibrium studies. The unfolding processes were monitored by circular dichroic and fluorescence spectroscopy. Experiments in the absence and presence of extraneous calcium in the concentration range 2 x 10(-3)-10(-1) M were performed. The presence of calcium in the weak calcium binding site changes the denaturation drastically. The heat- (or GnHCl-) induced unfolding curves obtained using CD spectroscopy show two independent transitions which seem not to have been resolved before. The presence of Ca2+ in the second (third in the case of thermitase) binding site increases the Tm values by 11-21 degrees C and the delta GD(H2O) values obtained from denaturation experiments in GnHCl by 6.7-7.2 kcal/mol when an extraneous Ca2+ concentration of 2 x 10(-2) M was used. One interpretation is that the initial step of denaturation in the presence of added calcium is the formation of a partially unfolded intermediate form, retaining a highly ordered structure with 60-85% of the alpha-helix structure of the native enzyme. This intermediate then unfolds at a temperature considerably higher than that of the same proteinases in the absence of added Ca2+. The free energy of stabilization of the intermediates is increased by 1.8-2.8 times in comparison with that for the unfolding reactions of the subtilases with empty Ca2/Ca3 binding sites. A second interpretation is that the two steps in the unfolding curves correspond to enzyme without and with calcium in the weak binding site. Fluorescence experiments confirm the mechanism involving the formation of intermediate states. The results are discussed in relation to the X-ray models of the six subtilases.

Binding Sites↗
PubMed1995

Linear and cyclic synthetic peptides related to the main autophosphorylation site of the Src tyrosine kinases as substrates and inhibitors of Lyn.

Tyrosine protein kinases (TPKs) of the src family contain two major phosphoacceptor sites which are homologous to the Tyr 416 and Tyr 527 of pp60c-src. The former represents the main autophosphorylation sites of these enzymes, and its phosphorylation correlates with increased kinase activity. It has previously been demonstrated that the Src-like tyrosine kinase expressed by the oncogene lyn displays a high affinity toward the heptapeptide H-Glu-Asp-Asn-Glu-Tyr-Thr-Ala-OH, which reproduces the main autophosphorylation site of the Src family enzymes [Donella-Deana, A., Marin, O., Brunati, A.M. & Pinna, L.A. (1992) Eur. J. Biochem. 204, 1159-1163]. Our study was addressed to the synthesis of some derivatives of this sequence in order to obtain both peptide substrates suitable for the detection of the Src-like tyrosine kinase activity and active site-directed inhibitors specific for this class of enzymes. For this purpose we synthesized by classical solution methods the heptapeptide and its dimeric form. Moreover, in order to improve the proteolytic resistance of these peptides we also synthesized their cyclic derivatives and their N-terminal acetylated and C-terminal amidated analogs. The correlation between the different structural properties induced by the modifications of the native sequence and the propensity of the peptides to act as Lyn substrates was examined. The kinetic data obtained indicate that the extent of the peptide phosphorylation varies considerably depending on the flexibility and length of the analogs. While the cyclization and the C-terminal amidation of the heptapeptide are detrimental for the Lyn activity, dimeric derivatives display very favourable kinetic constants. In particular the cyclic dimer is an especially suitable substrate for the tyrosine kinase and a powerful inhibitor of both the phosphorylation activity of Lyn and the enzyme autophosphorylation.

Amino Acid Sequence↗
PubMed1994

Activation of acetylcholinesterase by aluminium(III): the relevance of the metal species.

The present paper reports how aluminium [Al(III)] at a concentration of 3.7 microM can activate the bovine erythrocytic enzyme acetylcholinesterase (AChE) by about 38% in vitro. This same activating effect was observed on AChE form human as well as from rat erythrocyte ghosts and murine neuroblastoma cells. The interaction between Al3+ and gamma-peripheral sites of the enzyme produces AChE structural modifications as evidenced by circular dichroism measurements. This may provide a molecular explanation of the raised enzymatic activity.

Acetylcholinesterase↗
PubMed1994

Effects of beta 2-agonists during cardiopulmonary exercise test in COPD patients.

The aim of this study was to evaluate endocrine-metabolic, respiratory and cardiovascular effects of two beta 2-sympathomimetic selective agents, such as broxaterol and salbutamol, before and during cardiopulmonary exercise test (CPX). Twelve in-patients with chronic obstructive pulmonary disease (COPD) (with partially reversible airways obstruction) were included. Broxaterol (400 micrograms) and salbutamol (400 micrograms) were administered i.v., according to a double-blind, cross-over study. Before treatment and within 60 min after the administration of each agent, the patients underwent incremental CPX by bicycle ergometer to the maximum tolerable threshold. At these times the following variables were assessed: minute ventilation (VE), oxygen consumption (VO2), carbon dioxide production (VCO2), VE/VO2 ratio and O2 pulse, glycaemia, insulinaemia, plasma norepinephrine (NE) and epinephrine (E), arterial oxygen and carbon dioxide tension (PaO2 and PaCO2), plasma lactates, heart rate (HR), systolic (SBP) and diastolic (DBP) blood pressure. Spirometry was performed before and after the administration of each beta 2-adrenoceptor agonist. CPX brought about a significant increase in VE, VO2, VCO2, and O2 pulse. Broxaterol or salbutamol administration did not significantly modify the increases caused by CPX. At rest, 60 min after treatment, both bronchodilators caused a significant rise in glycaemia. A significant reduction of PaO2 (after broxaterol) and PaCO2 (after salbutamol) was observed at rest. In contrast, both agents caused no modification to potassium and insulin levels.(ABSTRACT TRUNCATED AT 250 WORDS)

Adolescent↗
PubMed1993

Synthesis and conformational studies on peptides corresponding to a putative autophosphorylation site of abl TPK.

The transforming gene of Abelson murine leukaemia virus (v-abl) codes for a membrane-associated tyrosine-specific protein kinase (abl TPK). Analysis of the v-abl gene has shown that both the fibroblast-transforming and tyrosine-protein kinase activities reside within a minimal region encoding a protein of 43 kDa (p43v-abl), which represents the most active, isolated form of this enzyme. Since the cellular substrates for p43v-abl are yet to be identified, we synthesized by classical solution methods the octapeptide H-Gly-Asp-Thr-Tyr-Thr-Ala-His-Ala-OH, corresponding to the structural sequence of the main putative autophosphorylation site (Tyr 515) of the abl TPK, as well as some of its analogs modified in positions -2, -1, +1 and +3. The synthetic peptides were tested as substrates for the p43v-abl. The kinetic data obtained indicate that the rates of their phosphorylation vary considerably depending on the sequence of the peptide, as expected. As a rule, no significant increment of the efficiency results from each substitution in the parent sequence. While the replacement of the two charged residues, namely Asp-2 and His-7, with neutral Ala is well tolerated, the substitution with amino acids bearing opposite charges is detrimental. The correlation between secondary structure of our synthetic octapeptides and their substrate recognition by p43v-abl was studied using CD and fluorescence spectroscopy in 5 mM Tris, in 98% TFE/Tris and in 30 mM SDS solutions. The comparison of the spectroscopic data with the kinetic parameters does not confirm a close relationship between the conformational properties of these peptides and their enzymatic role.

Amino Acid Sequence↗
PubMed1992

Spectroscopic studies on proteinase K and subtilisin DY. Relation to X-ray models.

Circular dichroic spectroscopy has been used to study the effect of pH, guanidinium hydrochloride concentration and temperature on the conformation of the fungal subtilisin-like proteinase K and the bacterial DY. The ellipticity of the bands in the far ultraviolet region remains almost unchanged in the pH range 3.0-11.0 (PMS-proteinase K) and 5.0-10.0 (PMS-subtilisin DY). The same ranges of pH stability were determined from the pH dependence of the near ultraviolet dichroic spectra. Hence the changes in the tertiary and secondary structure occur in parallel. Proteinase K is considerably more stable at acidic and somewhat more stable at alkaline pH than subtilisin DY. At neutral pH proteinase K is more resistant to denaturation by guanidinium hydrochloride than is subtilisin DY. The midpoints of the denaturation curves were 6.2 M and 3.2 M guanidinium, respectively. The thermal unfolding of proteinase K occurred at a higher temperature than for subtilisin DY, the transition midpoints being 65 degrees and 48 degrees, respectively. Thus proteinase K is overall a much more robust molecule than subtilisin DY, showing greater resistance to all three forms of denaturation. The differences in the stability of the two proteinases can be partly explained by differences in their calcium binding sites.

Circular Dichroism↗
PubMed1992

[Changes in the heart rhythm and slow-release beta-2 agonist therapy (peroral) in asthmatic patients. A Holter study].

Slow-release formulations of salbutamol, used in asthmatic patients, may have few effects on cardiac rhythm. This study was designed to compare incidence and type of cardiac arrhythmias in a group of ten patients with stable bronchial asthma before and after 6 weeks treatment with a slow-release formulation of salbutamol. After a baseline spirometric and electrocardiographic evaluation, patients received placebo for a two-weeks period. At the end of this period patients repeated spirometry and carried out a 24-hour-ambulatory-E-Holter-monitoring. Subsequently, patients received for 6 weeks a treatment with a slow-release formulation of oral salbutamol. At the end of this period patients repeated spirometry, a 24-hour-ambulatory-EG-Holter-monitoring and potassium serum levels were controlled. We concluded from our study that, in patients with moderate bronchial asthma the treatment with an oral formulation of slow-release salbutamol may carry out a significant and durable bronchodilator effect, without causing important cardiac arrhythmias.

Administration, Oral↗