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M Bolognesi

Publications and source records attributed to M Bolognesi.

At least 73 records · Page 4Linked to original sources
PubMed1998

Preliminary crystallographic investigations of recombinant GDP-4-keto-6-deoxy-D-mannose epimerase/reductase from E. coli.

The GDP-4-keto-6-deoxy-D-mannose epimerase/reductase (GM_ER) isolated from E. coli has been overexpressed as a GST-fusion protein and purified to homogeneity. The enzyme, an NADP+(H)-binding homodimer of 70 kDa, is responsible for the production of GDP-L-fucose. GM_ER shows significant structural homology to the human erythrocyte protein FX, which is involved in blood-group glycoconjugate biosynthesis, displaying 3,5 epimerase/reductase activity on GDP-4-keto-6-deoxy-D-mannose. GM_ER has been crystallized in a trigonal crystalline form, containing one molecule per asymmetric unit, suitable for high-resolution crystallographic investigations.

Amino Acid Sequence↗
PubMed1998

Double-stranded deoxyribonucleic acid binds to HLA class II molecules and inhibits HLA class II-mediated antigen presentation.

CD4+ T cells proliferating in response to purified double-stranded deoxyribonucleic acid (dsDNA) have been recently demonstrated in peripheral blood mononuclear cells of patients with systemic lupus erythematosus. Their activation was inhibited by anti-HLA class II (HLA-II) monoclonal antibodies; thus, the existence of a molecular interaction between dsDNA and HLA-II is conceivable. In this report we show that dsDNA specifically bind to HLA-II. After preincubating cells with purified dsDNA or synthetic oligonucleotides, dsDNA was detected on the cell membrane and in the lysates of HLA-II+ but not of isogenic HLA-II- cell lines. We demonstrate that dsDNA binding inhibits that of a specific peptide to HLA-II. Mixed lymphocyte reaction and antigen-specific T cell proliferation were inhibited by the preincubation of stimulator cells or antigen-presenting cells with dsDNA. These results suggest the existence of a novel mechanism of down-modulation of the CD4+ T cell function generated by lack of stimulation due to the HLA-II presenting molecules being "occupied" by dsDNA.

Animals↗
PubMed1998

Splenic impedance indices: a useful method to monitor patients after liver transplantation?

Splenic Doppler impedance indices are influenced, in portal hypertensive patients, by the resistance of the portal system. The aim of the study was to verify the usefulness of these indices in evaluating the presence of a pathological increase in portal resistance in patients with complications after liver transplantation. Splenic impedance indices have been evaluated in 46 patients before orthotopic liver transplantation (OLT), and 2 days, 1, 4, 8, and 12 to 18 months after transplantation. The results showed that spleen size slowly decreased after liver transplantation. From a baseline longitudinal diameter value of 18.0+/-3.6 cm (M+/-SD), the decrease was by 0%+/-3%, 8%+/-8%, 13%+/-9%, 15%+/-11%, and 14%+/-11% at 2 days and 1, 4, 8, and 12 to 18 months after liver transplantation. Splenic impedance indices-resistance index = (peak systolic - end diastolic) / peak systolic velocity; pulsatility index = (peak systolic - end diastolic) / mean velocity-which were increased before liver transplantation, showed a rapid decrease to normal values: resistance index: from 0.62+/-0.08 to 0.55+/-0.08 after 2 days, and to 0.49+/-0.09, 0.51+/-0.10, 0.54+/-0.10, 0.55+/-0.11 after 1, 4, 8, 12-18 months; pulsatility index: from 0.96+/-0.21 to 0.82+/-0.17 after 2 days, and to 0.69+/-0.19,0.72+/-0.21, 0.81+/-0.26, 0.84+/-0.26 after 1, 4, 8, and 12 to 18 months. Patients who had a good outcome, without any major complications, showed a clear and steady decrease in splenic impedance indices. On the contrary, patients who had complications affecting portal resistance (e.g., acute rejection, relapse of chronic hepatitis C virus-related hepatitis or cirrhosis, stenosis of portal anasthomosis, portal thrombosis), showed a lack of decrease, or, after an initial decrease, a subsequent re-increase in splenic impedance indices to pathological values. Splenic impedance indices measured in patients with complications were higher than those of patients without complications (P < .0004). Specificity and sensitivity of splenic impedance indices in the evaluation of the presence of complications increasing portal resistance were good. In conclusion, after OLT, splenic impedance indices could be useful aspecific parameters for identifying patients with complications that are able to affect or increase portal resistance.

Adult↗
PubMed1998

Hirunorms are true hirudin mimetics. The crystal structure of human alpha-thrombin-hirunorm V complex.

A novel class of synthetic, multisite-directed thrombin inhibitors, known as hirunorms, has been described recently. These compounds were designed to mimic the binding mode of hirudin, and they have been proven to be very strong and selective thrombin inhibitors. Here we report the crystal structure of the complex formed by human alpha-thrombin and hirunorm V, a 26-residue polypeptide containing non-natural amino acids, determined at 2.1 A resolution and refined to an R-factor of 0.176. The structure reveals that the inhibitor binding mode is distinctive of a true hirudin mimetic, and it highlights the molecular basis of the high inhibitory potency (Ki is in the picomolar range) and the strong selectivity of hirunorm V. Hirunorm V interacts through the N-terminal tetrapeptide with the thrombin active site in a nonsubstrate mode; at the same time, this inhibitor specifically binds through the C-terminal segment to the fibrinogen recognition exosite. The backbone of the N-terminal tetrapeptide Chg1"-Val2"-2-Nal3"-Thr4" (Chg, cyclohexyl-glycine; 2-Nal, beta-(2-naphthyl)-alanine) forms a short beta-strand parallel to thrombin main-chain residues Ser214-Gly219. The Chg1" side chain fills the S2 subsite, Val2" is located at the entrance of S1, whereas 2-Nal3" side chain occupies the aryl-binding site. Such backbone orientation is very close to that observed for the N-terminal residues of hirudin, and it is similar to that of the synthetic retro-binding peptide BMS-183507, but it is opposite to the proposed binding mode of fibrinogen and of small synthetic substrates. Hirunorm V C-terminal segment binds to the fibrinogen recognition exosite, similarly to what observed for hirudin C-termninal tail and related compounds. The linker polypeptide segment connecting hirunorm V N-and C-terminal regions is not observable in the electron density maps. The crystallographic analysis proves the correctness of the design and it provides a compelling proof on the interaction mechanism for this novel class of high potency multisite-directed synthetic thrombin inhibitors.

Antithrombins↗
PubMed1998

Doppler sonography and hepatic vein catheterization in portal hypertension: assessment of agreement in evaluating severity and response to treatment.

BACKGROUND/AIM: The study was designed to assess to what extent portal hemodynamic parameters obtained by duplex Doppler ultrasonography may be substituted for the measurement of hepatic venous pressure gradient in evaluating the severity of portal hypertension and the response to medical treatment with beta-blockers or beta-blockers plus nitrates in patients with cirrhosis and portal hypertension. METHODS: In 39 of these patients hepatic venous pressure gradient was determined by hepatic vein catheterization, and portal blood flow velocity and the congestion index of the portal vein were measured by duplex Doppler ultrasonography. In 19 of these patients the changes in hepatic venous pressure gradient and in Doppler parameters were also assessed after chronic administration of nadolol. In 11 of the 19 patients the changes after chronic administration of nadolol plus isosorbide-5-mononitrate were also measured. RESULTS: In the whole series, no significant correlation was found between hepatic venous pressure gradient and duplex Doppler parameters, but, when the 12 patients with a patent para-umbilical vein were excluded, significant linear correlations were found between hepatic venous pressure gradient and portal blood velocity (r=-0.39; p=0.05) or congestion index (r=0.37; p=0.05). Considering together the changes induced by nadolol and nadolol plus isosorbide-5-mononitrate, no correlation was apparent between changes in duplex Doppler parameters and in hepatic venous pressure gradient. Agreement between hepatic venous pressure gradient and duplex Doppler parameters in defining good and poor responders was insufficient. CONCLUSIONS: These data suggest that portal blood velocity and the congestion index of the portal vein are related to portal hypertension in patients without a patent para-umbilical vein, but are of limited value in discriminating good responders from poor responders to medical treatment for portal hypertension.

Adrenergic beta-Antagonists↗
PubMed1998

Cross-enzyme inhibition by gabexate mesylate: formulation and reactivity study.

Gabexate mesylate (GM; commercialized under the brand name FOY) is a nonantigenic synthetic inhibitor of plasmatic and pancreatic serine proteinases that is used therapeutically in the treatment of pancreatitis and disseminated intravascular coagulation and as a regional anticoagulant for hemodialysis. The inhibitory effect of GM on nitric oxide synthase as well as serine proteinases and swine kidney copper amine oxidase, all acting on cationic substrates, has been investigated. On the basis of the available X-ray crystal structures of the enzymes considered, the possible binding mode(s) of GM has(have) been analyzed. The enzyme cross-inhibition by GM suggests that the use of this drug should be under careful control. With the aim to improve the scarce plasma stability of GM, the positively charged drug has been complexed to the surface of preformed anionic liposomes. The liposome-complexed GM half-life increases about five-fold, indicating the protective effect of liposomes on GM degradation. Moreover, the GM complexation with liposomes does not alter its inhibitory activity on NOS-I and porcine pancreatic trypsin.

Drug Carriers↗
PubMed1998

Updating prognosis of cirrhosis by Cox's regression model using Child-Pugh score and aminopyrine breath test as time-dependent covariates.

BACKGROUND AND AIMS: The determination of aminopyrine breath test on entry into the study was recently shown to improve the accuracy of prediction of death based on the Child-Pugh classification, but the possible usefulness of serial determinations of both parameters has not been assessed. In the present study, we aimed at evaluating whether serial determinations of aminopyrine breath test and Child-Pugh score improve prognostic accuracy in patients with cirrhosis, compared with determinations obtained only on admission. PATIENTS: In 74 patients with liver cirrhosis aminopyrine breath test and Child-Pugh score were obtained upon entry into the study. Patients were followed with sequential aminopyrine breath tests and assessments of the Child-Pugh score every 4-6 months. A total number of 232 determinations were obtained. During follow-up 45 patients died, on average after 12 months of follow-up. RESULTS: Child-Pugh score improved in the beginning of follow-up, and then remained fairly constant; aminopyrine breath test showed no improvement in the beginning of follow-up, but rather a slowly progressive decline. In patients who died, both the Child-Pugh score and the metabolism of aminopyrine were significantly more impaired in the last year preceding death (p < 0.05). Applying Cox's regression model with time-dependent covariates, Child-Pugh score and aminopyrine breath test were independent significant predictors of survival. The model with time-dependent covariates explained the observed survival much better than the model with time-fixed covariates (chi-sq. explained by regression = 31.45 vs 11.97; d.f. = 2; p = 0.0000001 vs 0.003). CONCLUSIONS: These data suggest that serial determinations of Child-Pugh score and aminopyrine breath test can be used to efficiently update prognosis of cirrhosis.

Aminopyrine↗
PubMed1998

Leech antihemostatic proteins share the T-knot scaffold, a disulfide-reinforced structural motif.

The occurrence of similar topologies among unrelated proteins is an emerging theme in structural biology. Here we report that the T-knot scaffold, a disulfide-reinforced structural motif shared by knottins and EGF-like proteins, is also present in leech antihemostatic proteins. Our finding emphasizes the versatile nature of this small structural motif, representing a compact structural unit suitable for the diverse biological functions performed by knottins, EGF-like proteins and leech antihemostatic proteins.

Animals↗
PubMed1997

Evidence of his61 imidazolate bridge rupture in reduced crystalline Cu,Zn superoxide dismutase.

X-ray absorption spectroscopy has been carried out on the copper K edge in oxidized and reduced bovine Cu,Zn SOD in solution and in crystalline state. The results indicate that the copper coordination geometry is unaffected by the solution or by the crystalline state of the protein, in both oxidation states. Moreover the two oxidation states of the active copper ion are reflected under, all the experimental conditions, by distinct coordination spheres around the catalytic metal, which is four-coordinated and three-coordinated in the Cu(II) and in the Cu(I) enzyme, respectively.

Absorptiometry, Photon↗
PubMed1997

Unique structural features of the monomeric Cu,Zn superoxide dismutase from Escherichia coli, revealed by X-ray crystallography.

The first three-dimensional structure of a functional monomeric Cu, Zn superoxide dismutase (from Escherichia coli, E_SOD) is reported at 2.0 A resolution (R-factor=16.8%). Compared to the homologous eukaryotic enzymes, E_SOD displays a perturbed antiparallel beta-barrel structure. The most striking structural features observed include extended amino acid insertions in the surface 1, 2-loop and S-S subloop, modification of the disulfide bridge connection, and loss of functional electrostatic residues, suggesting a modified control of substrate steering toward the catalytic center. The active site Cu2+ displays a distorted coordination sphere due to an unusually long bond to the metal-bridging residue His61. Inspection of the crystal packing does not show regions of extended contact indicative of a dimeric assembly. The molecular surface region involved in subunit dimerization in eukaryotic superoxide dismutases is structurally altered in E_SOD and displays a net polar nature.

Amino Acid Sequence↗
PubMed1997

Human alpha-thrombin inhibition by the highly selective compounds N-ethoxycarbonyl-D-Phe-Pro-alpha-azaLys p-nitrophenyl ester and N-carbobenzoxy-Pro-alpha-azaLys p-nitrophenyl ester: a kinetic, thermodynamic and X-ray crystallographic study.

Kinetics, thermodynamics and structural aspects of human alpha-thrombin (thrombin) inhibition by newly synthesized low molecular weight derivatives of alpha-azalysine have been investigated. The thrombin catalyzed hydrolysis of N-ethoxycarbonyl-D-Phe-Pro-alpha-azaLys p-nitrophenyl ester (Eoc-D-Phe-Pro-azaLys-ONp) and N-carbobenzoxy-Pro-alpha-azaLys p-nitrophenyl ester (Cbz-Pro-azaLys-ONp) was investigated at pH 6.2 and 21.0 degrees C, and analyzed in parallel with that of N-alpha-(N,N-dimethylcarbamoyl)-alpha-azalysine p-nitrophenyl ester (Dmc-azaLys-ONp). Decarboxylation following the enzymatic hydrolysis of these p-nitrophenyl esters gave the corresponding 1-peptidyl-2(4-aminobutyl) hydrazines (peptidyl-Abh) showing properties of thrombin competitive inhibitors. Therefore, thermodynamics for the reversible binding of D-Phe-Pro-Abh, Cbz-Pro-Abh and Dmc-Abh to thrombin was examined. These results are consistent with the minimum four-step catalytic mechanism for product inhibition of serine proteinases. Eoc-D-Phe-Pro-azaLys-ONp and Eoc-D-Phe-Pro-Abh display a sub-micromolar affinity for thrombin together with a high selectivity versus homologous plasmatic and pancreatic serine proteinases acting on cationic substrates. The three-dimensional structures of the reversible non-covalent thrombin:Eoc-D-Phe-Pro-Abh and thrombin:Cbz-Pro-Abh complexes have been determined by X-ray crystallography at 2.0 A resolution (R-factor = 0.169 and 0.179, respectively), and analyzed in parallel with that of the thrombin:Dmc-azaLys acyl-enzyme adduct. Both Eoc-D-Phe-Pro-Abh and Cbz-Pro-Abh competitive inhibitors are accommodated in the thrombin active center, spanning the region between the aryl binding site and the S1 primary specificity subsite.

Animals↗
PubMed1997

Active site plasticity in D-amino acid oxidase: a crystallographic analysis.

D-Amino acid oxidase (DAAO) is the prototype of the flavin-containing oxidases. It catalyzes the oxidative deamination of various D-amino acids, ranging from D-Ala to D-Trp. We have carried out the X-ray analysis of reduced DAAO in complex with the reaction product imino tryptophan (iTrp) and of the covalent adduct generated by the photoinduced reaction of the flavin with 3-methyl-2-oxobutyric acid (kVal). These structures were solved by combination of 8-fold density averaging and least-squares refinement techniques. The FAD redox state of DAAO crystals was assessed by single-crystal polarized absorption microspectrophotometry. iTrp binds to the reduced enzyme with the N, C alpha, C, and C beta atoms positioned 3.8 A from the re side of the flavin. The indole side chain points away from the cofactor and is bound in the active site through a rotation of Tyr224. This residue plays a crucial role in that it adapts its conformation to the size of the active site ligand, providing the enzyme with the plasticity required for binding a broad range of substrates. The iTrp binding mode is fully consistent with the proposal, inferred from the analysis of the native DAAO structure, that substrate oxidation occurs via direct hydride transfer from the C alpha to the flavin N5 atom. In this regard, it is remarkable that, even in the presence of the bulky iTrp ligand, the active center is made solvent inaccessible by loop 216-228. This loop is thought to switch between the "closed" conformation observed in the crystal structures and an "open" state required for substrate binding and product release. Loop closure is likely to have a role in catalysis by increasing the hydrophobicity of the active site, thus making the hydride transfer reaction more effective. Binding of kVal leads to keto acid decarboxylation and formation of a covalent bond between the keto acid C alpha and the flavin N5 atoms. Formation of this acyl adduct results in a nonplanar flavin, characterized by a 22 degrees angle between the pyrimidine and benzene rings. Thus, in addition to an adaptable substrate binding site, DAAO has the ability to bind a highly distorted cofactor. This ability is relevant for the enzyme's function as a highly efficient oxidase.

Arginine↗
PubMed1997

pH dependence of bovine mast cell tryptase catalytic activity and of its inhibition by 4',6-diamidino-2-phenylindole.

Tryptases are oligomeric enzymes localized in the secretory granules of mast cells. Their role(s) in vivo has yet to be clarified and the lack of powerful and specific inhibitors has hampered the comprehension of the biological functions of these enzymes. In this paper, we identify 4',6-diamidino-2-phenylindole as a potent inhibitor for bovine tryptase. This inhibitory effect and the enzyme catalyzed hydrolysis of the synthetic substrate Boc-Phe-Ser-Arg-methyl-coumarin were investigated in the pH range of 6.0-9.0. On the basis of the pK shifts occurring upon formation of the inhibitor(substrate)/enzyme complexes, some aminoacidic groups are proposed to play a role in such interactions.

Animals↗
PubMed1997

Unusual structure of the oxygen-binding site in the dimeric bacterial hemoglobin from Vitreoscilla sp.

BACKGROUND: The first hemoglobin identified in bacteria was isolated from Vitreoscilla stercoraria (VtHb) as a homodimeric species. The wild-type protein has been reported to display medium oxygen affinity and cooperative ligand-binding properties. Moreover, VtHb can support aerobic growth in Escherichia coli with impaired terminal oxidase function. This ability of VtHb to improve the growth properties of E. coli has important applications in fermentation technology, assisting the overexpression of recombinant proteins and antibiotics. Oxygen binding heme domains have been identified in chimeric proteins from bacteria and yeast, where they are covalently linked to FAD- and NAD(P)H-binding domains. We investigate here the fold, the distal heme site structure and the quaternary assembly of a bacterial hemoglobin which does not bear the typical flavohemoglobin domain organization. RESULTS: The VtHb three-dimensional structure conforms to the well known globin fold. Nevertheless, the polypeptide segment connecting helices C and E is disordered, and residues E7-E10 (defined according to the standard globin fold nomenclature) do not adopt the usual alpha-helical conformation, thus locating Gln53(E7) out of the heme pocket. Binding of azide to the heme iron introduces substantial structural perturbations in the heme distal site residues, particularly Tyr29(B10) and Pro54(E8). The quaternary assembly of homodimeric VtHb, not observed before within the globin family, is based on a molecular interface defined by helices F and H of both subunits, the two heme iron atoms being 34 A apart. CONCLUSIONS: The unusual heme distal site structure observed shows that previously undescribed molecular mechanisms of ligand stabilization are operative in VtHb. The polypeptide chain disorder observed in the CE region indicates a potential site of interaction with the FAD/NADH reductase partner, in analogy with observations in the chimeric flavohemoglobin from Alcaligenes eutrophus.

Amino Acid Sequence↗
PubMed1997

Determining mutational fingerprints at the human p53 locus with a yeast functional assay: a new tool for molecular epidemiology.

In order to isolate experimentally induced p53 mutations, a yeast expression vector harbouring a human wild-type p53 cDNA was treated in vitro with the antineoplastic drug chloroethyl-cyclohexyl-nitroso-urea (CCNU) and transfected into a yeast strain containing the ADE2 gene regulated by a p53-responsive promoter. p53 mutations were identified in 32 out of 39 plasmids rescued from independent ade- transformants. Ninety-two percent of CCNU induced mutations were GC-targeted single base pair substitutions, and GC > AT transitions represented 73% of all single base pair substitutions. In 70% of the cases the mutated G was preceded 5' by a purine. The distribution of the mutations along the p53 cDNA was not random: positions 734 and 785 appeared as CCNU mutational hotspots (n=3, P<0.0003) and CCNU induced only GC > AT transitions at those positions. The features of these CCNU-induced mutations are consistent with the hypothesis that O6-alkylguanine is the major causative lesion. One third of the CCNU-induced mutants were absent from a huge collection of 4496 p53 mutations in human tumours and cell lines, thus demonstrating that CCNU has a mutational spectrum which is uniquely different from that of naturally selected mutations. This strategy allows direct comparison of observed natural mutation spectra with experimentally induced mutation spectra and opens the way to a more rigorous approach in the field of molecular epidemiology.

Antineoplastic Agents, Alkylating↗
PubMed1997

Expression, purification, crystallization, and preliminary X-ray diffraction analysis of the homodimeric bacterial hemoglobin from Vitreoscilla stercoraria.

The recombinant homodimeric hemoglobin from the strictly aerobe gram-negative bacterium Vitreoscilla stercoraria has been expressed in Escherichia coli, purified to homogeneity, and crystallized by vapor diffusion techniques, using ammonium sulfate as precipitant. The crystals belong to the monoclinic space group P2(1) and diffract to HIGH resolution. The unit cell parameters are alpha = 62.9, b = 42.5, c = 63.2 A, beta = 106.6 degrees; the asymmetric unit contains the homodimeric hemoglobin, with a volume solvent content of 42%.

Bacillus subtilis↗
PubMed1997

Effects of chronic therapy with nadolol on portal hemodynamics and on splanchnic impedance indices using Doppler sonography: comparison between acute and chronic effects.

BACKGROUND/AIMS: Beta-blockers are currently used for chronic therapy of portal hypertension. Duplex Doppler ultrasonography has been proposed for non-invasive evaluation of splanchnic pharmacodynamics, but the chronic effects of beta-blockers on portal hemodynamics and on splanchnic arterial impedance indices have not been analyzed with this method. This was the aim of the study. METHODS: The effects of acute (80 mg p.o.) and chronic (2 months at a dosage sufficient to reduce heart rate by at least 25% in respect of basal values) nadolol administration on portal blood flow velocity and volume, and on splanchnic and renal arterial impedance indices [pulsatility index = (peak systolic velocity-minimum velocity)/mean velocity] were evaluated in patients with cirrhosis. Twenty-eight patients with cirrhosis and portal hypertension were investigated. Nineteen patients received nadolol, and nine received placebo. RESULTS: Placebo caused no significant hemodynamic change. Portal blood flow mean velocity decreased after chronic therapy (11.7 +/- 2.9 cm/s to 9.1 +/- 2.3, p < 0.001). In the 16 patients with acute and chronic evaluation, portal blood flow mean velocity decreased after acute therapy (11.8 +/- 3.0 cm/s to 10.4 +/- 3.0, p < 0.01), and even more so after chronic therapy (to 9.2 +/- 2.4, p < 0.01). No parallel was found between acute and chronic effects. Hepatic, mesenteric and splenic pulsatility indices increased after chronic therapy (1.26 +/- 0.33 to 1.39 +/- 0.28, p < 0.02; 2.04 +/- 0.41 to 2.50 +/- 0.61, p < 0.01; 0.92 +/- 0.22 to 1.18 +/- 0.27, p < 0.001 respectively); renal pulsatility index increased (1.12 +/- 0.20 to 1.40 +/- 0.28, p < 0.001). CONCLUSIONS: Chronic therapy with nadolol decreased portal blood flow velocity and volume, and increased splanchnic and renal impedance indices. Chronic effects of nadolol on portal inflow cannot be predicted from its acute effects. Evaluation of the effect of nadolol on portal blood velocity and volume should be performed after chronic therapy. Duplex Doppler ultrasonography allows venous and arterial splanchnic pharmacodynamics to be studied separately.

Adrenergic beta-Antagonists↗