Biomedical subjects

M Bolognesi

Publications and source records attributed to M Bolognesi.

At least 19 recordsLinked to original sources

PubMed2005

Heme oxygenase-1 transduction in endothelial cells causes downregulation of monocyte chemoattractant protein-1 and of genes involved in inflammation and growth.

Heme oxygenase (HO-1) has been implicated as an anti-inflammatory gene. HO-1 overexpression, transiently and chronically, affects heme protein expression, attenuates TNF-mediated cell death, and decreases adhesion molecules. We assessed the effect of oxidant-mediated agents such as glucose and heme on 8-epi-isoprostane PGF2alpha (8-epi-PGF2alpha) and monocyte chemoattractant protein-1 (MCP-1). Glucose and heme increased both 8-epi-PGF2alpha and MCP-1. Overexpression of HO-1 decreased both 8-epi-PGF2alpha and MCP-1. To identify target genes involved in HO-1-mediated regulation of inflammation, a serial analysis of gene expression mRNA profile was performed in endothelial cells (EC) overexpressing the human HO-1 gene by transduction of a retrovirus carrying the HO-1 gene. Gene arrays (differential displays among 2400 genes) were used to identify known and novel differentially expressed genes. The levels of expression for several genes were confirmed by real time PCR in cells overexpressing the HO-1 gene. In HO-1 overexpressing cells, VEGF and the prostaglandin transporter were greatly increased while MCP-1 levels were decreased by 2.5-fold. The data from this study are relevant to understanding the mechanisms underlying the pathophysiological effects of HO-1 deficiency on endothelial cell injury and inflammation.

Cell Cycle↗

PubMed2005

Disagreement between acute and chronic haemodynamic effects of nadolol in cirrhosis: a pathophysiological interpretation.

BACKGROUND: The acute effects of beta-blockers may be different from chronic; mechanisms underlying this difference are poorly elucidated. AIM: To assess portal pressure and its pathophysiological determinants after acute and chronic administration of nadolol. METHODS: In 24 patients with cirrhosis and portal hypertension hepatic venous pressure gradient, portal blood flow and resistance to portal blood flow were measured before, 60-90 min after acute administration of nadolol, and after 1 month. Patients were good-responders if hepatic venous pressure gradient was < or =12 mmHg, or decreased by at least 20%. RESULTS: Eleven and 13 patients were good- and poor-responders to acute administration, respectively. Acute poor-responders showed a lower decrease in portal blood flow (P = 0.04) and a less evident decrease in mean arterial pressure (P < 0.001). Eleven and 13 patients were good- and poor-responders to chronic administration, respectively. Chronic poor-responders showed a larger increase in resistance to portal blood flow compared with good-responders (P = 0.01). Disagreement between acute and chronic effects was seen in 12 patients: six were acute good-responders chronic poor-responders and six were acute poor-responders chronic good-responders. Acute good-responders chronic poor-responders patients had the smallest decreases in portal blood flow and in mean arterial pressure after acute administration, while acute poor-responders chronic good-responders showed the largest (P = 0.05 and 0.01). CONCLUSIONS: Disagreement between acute and chronic effects of nadolol on hepatic venous pressure gradient is common. The mechanism responsible is complex, the acute effect being mainly modulated by arterial hypotension and the chronic effect by changes in portal resistance.

Acute Disease↗

PubMed2005

Haeme oxygenase mediates hyporeactivity to phenylephrine in the mesenteric vessels of cirrhotic rats with ascites.

BACKGROUND AND AIMS: Haeme oxygenase could play a role in the pathogenesis of arterial vasodilation in cirrhosis. The aim of this study was to verify the role of haeme oxygenase in the hyporesponsiveness to phenylephrine of small mesenteric arteries in rats with CCl(4) induced cirrhosis, with and without ascites. METHODS: Pressurised small resistance mesenteric arteries were challenged with increasing doses of phenylephrine. Dose-response curves were evaluated under basal conditions, after inhibition of haeme oxygenase with chromium-mesoporphyrin, after inhibition of nitric oxide synthase (NOS) with N(G)-nitro-L-arginine-methyl-ester (L-NAME), and then after inhibition of both NOS and haeme oxygenase. Haeme oxygenase protein expression was also analysed. RESULTS: Twenty six control rats and 35 rats with cirrhosis (17 with and 18 without ascites) were studied. Response to phenylephrine was lower in non-ascitic and ascitic cirrhosis than in controls. Chromium-mesoporphyrin increased the response to phenylephrine only in ascitic cirrhosis (p<0.001). L-NAME increased the response to phenylephrine in controls (p<0.001) and in ascitic and non-ascitic cirrhosis (p = 0.002, p<0.001, respectively) but the final response in non-ascitic cirrhosis was similar to that of control rats while it remained impaired in ascitic cirrhosis. Addition of chromium-mesoporphyrin to L-NAME improved the response to phenylephrine in ascitic cirrhosis (p<0.01), with final values not different from those of the other two groups. Protein expression of the inducible isoform of haeme oxygenase was increased in the mesenteric vessels of cirrhotic rats. CONCLUSION: Haeme oxygenase mediates hyporeactivity to phenylephrine in the mesenteric vessels of experimental cirrhosis with ascites. NOS plays a major role only in the first stage of the disease.

Animals↗

PubMed2004

beta2-microglobulin H31Y variant 3D structure highlights the protein natural propensity towards intermolecular aggregation.

beta2-Microglobulin (beta2m) is the non-covalently bound light chain of the human class I major histocompatibility complex (MHC-I). The natural turnover of MHC-I gives rise to the release of beta2m into plasmatic fluids and to its catabolism in the kidney. beta2m dissociation from the heavy chain of the complex is a severe complication in patients receiving prolonged hemodialysis. As a consequence of renal failure, the increasing beta2m concentrations can lead to deposition of the protein as amyloid fibrils. Here we characterize the His31-->Tyr human beta2m mutant, a non-natural form of beta2m that is more stable than the wild-type protein, displaying a ten-fold acceleration of the slow phase of folding. We report the 2.9A resolution crystal structure and the NMR characterization of the mutant beta2m, focussing on selected structural features and on the molecular packing observed in the crystals. Juxtaposition of the four mutant beta2m molecules contained in the crystal asymmetric unit, and specific hydrogen bonds, stabilize a compact protein assembly. Conformational heterogeneity of the four independent molecules, some of their mutual interactions and partial unpairing of the N-terminal beta-strand in one protomer are in keeping with the amyloidogenic properties displayed by the mutant beta2m.

Amino Acid Substitution↗

PubMed2003

Static and dynamic water molecules in Cu,Zn superoxide dismutase.

Understanding protein hydration is a crucial, and often underestimated issue, in unraveling protein function. Molecular dynamics (MD) computer simulation can provide a microscopic description of the water behavior. We have applied such a simulative approach to dimeric Photobacterium leiognathi Cu,Zn superoxide dismutase, comparing the water molecule sites determined using 1.0 ns MD simulation with those detected by X-ray crystallography. Of the water molecules detected by the two techniques, 20% fall at common sites. These are evenly distributed over the protein surface and located around crevices, which represent the preferred hydration sites. The water mean residence time, estimated by means of a survival probability function on a given protein hydration shell, is relatively short and increases for low accessibility sites constituted by polar atoms. Water molecules trapped in the dimeric protein intersubunit cavity, as identified in the crystal structure, display a trajectory mainly confined within the cavity. The simulation shows that these water molecules are characterized by relatively short residence times, because they continuously change from one site to another within the cavity, thus hinting at the absence of any relationship between spatial and temporal order for solvent molecules in proximity of protein surface.

Algorithms↗

PubMed2003

Active-site copper and zinc ions modulate the quaternary structure of prokaryotic Cu,Zn superoxide dismutase.

The influence of the constitutive metal ions on the equilibrium properties of dimeric Photobacterium leiognathi Cu,Zn superoxide dismutase has been studied for the wild-type and for two mutant protein forms bearing a negative charge in the amino acid clusters at the dimer association interface. Depletion of copper and zinc dissociates the two mutant proteins into monomers, which reassemble toward the dimeric state upon addition of stoichiometric amounts of zinc. Pressure-dependent dissociation is observed for the copper-depleted wild-type and mutated enzymes, as monitored by the fluorescence shift of a unique tryptophan residue located at the subunit association interface. The spectral shift occurs slowly, reaching a plateau after 15-20 minutes, and is fully reversible. The recovery of the original fluorescence properties, after decompression, is fast (less than four minutes), suggesting that the isolated subunit has a relatively stable structure, and excluding the presence of stable intermediates during the dimer-monomer transition. The dimer dissociation process is still incomplete at 6.5 kbar for the copper-depleted wild-type and mutated enzymes, at variance with what is generally observed for oligomeric proteins that dissociate below 3 kbar. Measurement of the degree of dissociation, at two different protein concentrations, allows us to calculate the standard volume variation upon association, Delta V, and the dissociation constant K(d0), at atmospheric pressure, (25 ml/mol and 3 x 10(-7)M, respectively). The holoprotein is fully dimeric even at 6.5 kbar, which allows us to evaluate a lower Delta G degrees limit of 11.5 kcal/mol, corresponding to a dissociation constant K(d0)<10(-9)M.

Binding Sites↗

PubMed2003

Portal hypertension and portal hypertensive gastropathy in patients with liver cirrhosis: a haemodynamic study.

BACKGROUND/AIM: The relationships between the levels of portal hypertension and the morphologic alterations of gastric mucosa in patients with liver cirrhosis--generally described as portal hypertensive gastropathy--are poorly defined. PATIENTS: In total, 62 patients with cirrhosis of different aetiologies, were examined by endoscopy and measurement of portal hypertension by hepatic venous pressure gradient. RESULTS: Portal hypertensive gastropathy was observed in 49 cases; six patients showed gastric antral vascular ectasia always associated with gastric lesions described as severe portal hypertensive gastropathy with different localizations. Hepatic venous pressure gradient showed severe portal hypertension in 37 cases, and averaged 17.7 +/- 4.3 mmHg. It was much higher in patients with severe lesions (p=0.0004). Hepatic venous pressure gradient in patients with endoscopic signs of isolated antral gastropathy was lower (p=0.04) than in those with isolated lesions in body-fundus. No relationship was found between hepatic function, as assessed by the Child-Pugh score, and portal hypertensive gastropathy. CONCLUSIONS: The present data suggest that the severity of portal hypertensive gastropathy is related to portal hypertension, but portal hypertension is not the sole determinant of the occurrence of endoscopic abnormalities of gastric mucosa. The derangement of liver function does not appear to play any role in the occurrence of portal hypertensive gastropathy.

Endoscopy, Digestive System↗

PubMed2002

Role of spleen enlargement in cirrhosis with portal hypertension.

The possible relationships between splenomegaly and portal hypertension have been analysed in patients with cirrhosis. In this condition, splenomegaly is not only caused by portal congestion, but it is mainly due to tissue hyperplasia and fibrosis. The increase in spleen size is followed by an increase in splenic blood flow, which participates in portal hypertension actively congesting the portal system.

Endothelins↗

PubMed2001

Crystallization and preliminary X-ray analysis of neural haemoglobin from the nemertean worm Cerebratulus lacteus.

The nemertean worm Cerebratulus lacteus neural tissue haemoglobin (109 amino acids, the shortest known haemoglobin) has been overexpressed in Escherichia coli, purified and crystallized. A highly redundant native data set has been collected at the Cu K(alpha) wavelength to 2.05 A resolution. The crystals belong to the orthorhombic P2(1)2(1)2(1) space group, with unit-cell parameters a = 42.5, b = 43.1, c = 60.2 A and one molecule per asymmetric unit. The anomalous difference Patterson map clearly reveals the position of the haem Fe atom, thus paving the way for MAD/SAD structure determination.

Animals↗

PubMed2001

Bone marrow transplantation from unrelated donors: the impact of mismatches with substitutions at position 116 of the human leukocyte antigen class I heavy chain.

The hypothesis was tested that amino acid substitutions in specific positions within human leukocyte antigen class I heavy chain would have different impacts on transplant-related mortality (TRM) in patients receiving transplanted bone marrow from unrelated donors. One hundred patients and their unrelated donors were typed by sequence-based typing for the human leukocyte antigen (HLA)-A, -B, and -C loci. All pairs were matched for DRB1, DRB3, DRB4, DRB5, DQA1, and DQB1 loci. Forty pairs were also matched at class I, and 60 pairs had one or more mismatches at class I loci. It was found that substitutions at positions 116 and 114 of class I heavy chain significantly increased the risk for TRM in univariate and bivariate Cox analyses. Conversely, no association between number of multiple mismatches or number of amino acid substitutions and TRM was seen when positions 116 and 114 were adjusted for. Variables predictive of TRM in multivariate Cox analysis were number of cells infused, diagnosis (chronic myeloid leukemia [CML] or non-CML), and amino acid substitution at position 116 or 152. The only variable predictive of severe acute graft-versus-host disease (GVHD) in multivariate Cox analysis was substitution at position 116. Actuarial risk for acute GVHD grade III-IV, TRM, and relapse in pairs with substitutions at position 116 (n = 37) compared to other pairs (n = 63) was, respectively, 36% versus 14% (P =.01), 59% versus 28% (P =.001), and 25% versus 31% (P =.4). In conclusion these data suggest that substitutions at position 116 of class I heavy chain increase the risk for acute GVHD and TRM in patients who receive transplanted bone marrow from unrelated donors.

Adult↗

PubMed2001

Kinetic and crystallographic analyses support a sequential-ordered bi bi catalytic mechanism for Escherichia coli glucose-1-phosphate thymidylyltransferase.

Glucose-1-phosphate thymidylyltransferase is the first enzyme in the biosynthesis of dTDP-l-rhamnose, the precursor of l-rhamnose, an essential component of surface antigens, such as the O-lipopolysaccharide, mediating virulence and adhesion to host tissues in many microorganisms. The enzyme catalyses the formation of dTDP-glucose, from dTTP and glucose 1-phosphate, as well as its pyrophosphorolysis. To shed more light on the catalytic properties of glucose-1-phosphate thymidylyltransferase from Escherichia coli, specifically distinguishing between ping pong and sequential ordered bi bi reaction mechanisms, the enzyme kinetic properties have been analysed in the presence of different substrates and inhibitors. Moreover, three different complexes of glucose-1-phosphate thymidylyltransferase (co-crystallized with dTDP, with dTMP and glucose-1-phosphate, with d-thymidine and glucose-1-phosphate) have been analysed by X-ray crystallography, in the 1.9-2.3 A resolution range (R-factors of 17.3-17.5 %). The homotetrameric enzyme shows strongly conserved substrate/inhibitor binding modes in a surface cavity next to the topological switch-point of a quasi-Rossmann fold. Inspection of the subunit tertiary structure reveals relationships to other enzymes involved in the biosynthesis of nucleotide-sugars, including distant proteins such as the molybdenum cofactor biosynthesis protein MobA. The precise location of the substrate relative to putative reactive residues in the catalytic center suggests that, in keeping with the results of the kinetic measurements, both catalysed reactions, i.e. dTDP-glucose biosynthesis and pyrophosphorolysis, follow a sequential ordered bi bi catalytic mechanism.

Amino Acid Sequence↗

PubMed2001

The 1.6 A resolution crystal structure of a mutant plastocyanin bearing a 21-25 engineered disulfide bridge.

Plastocyanin is an electron-transfer protein which has been largely used for biophysical studies as well as for protein-engineering experiments. A surface disulfide bridge has been engineered in poplar plastocyanin to allow protein chemisorption on gold substrates. The mutated plastocyanin crystal structure has been studied at 1.6 A resolution (R factor = 0.145, R(free) = 0.205) to characterize the effects of the engineered disulfide on the overall protein structure and on the Cu-coordination sphere in view of biophysical applications. The new orthorhombic crystal form isolated for the mutated plastocyanin displays two protein molecules per asymmetric unit.

Crystallization↗

PubMed2001

Monomer-dimer equilibrium and oxygen binding properties of ferrous Vitreoscilla hemoglobin.

The monomer-dimer equilibrium and the oxygen binding properties of ferrous recombinant Vitreoscilla hemoglobin (Vitreoscilla Hb) have been investigated. Sedimentation equilibrium data indicate that the ferrous deoxygenated and carbonylated derivatives display low values of equilibrium dimerization constants, 6 x 10(2) and 1 x 10(2) M(-1), respectively, at pH 7.0 and 10 degrees C. The behavior of the oxygenated species, as measured in sedimentation velocity experiments, is superimposable to that of the carbonylated derivative. The kinetics of O(2) combination, measured by laser photolysis at pH 7.0 and 20 degrees C, is characterized by a second-order rate constant of 2 x 10(8) M(-1) s(-1) whereas the kinetics of O(2) release at pH 7.0 is biphasic between 10 and 40 degrees C, becoming essentially monophasic below 10 degrees C. Values of the first-order rate constants (at 20 degrees C) and of the activation energies for the fast and slow phases of the Vitreoscilla Hb deoxygenation process are 4.2 s(-1) and 19.2 kcal mol(-1) and 0.15 s(-1) and 24.8 kcal mol(-1), respectively. Thus the biphasic kinetics of Vitreoscilla Hb deoxygenation is unrelated to the association state of the protein. The observed biphasic oxygen release may be accounted for by the presence of two different conformers in thermal equilibrium within the monomer. The two conformers may be assigned to a structure in which the heme-iron-bound ligand is stabilized by direct hydrogen bonding to TyrB10 and a structure in which such interaction is absent. The slow interconversion between the two conformers may reflect a very large conformational rearrangement in the disordered distal pocket segment connecting helices C and E.

Bacterial Proteins↗

PubMed2001

Mycobacterium tuberculosis hemoglobin N displays a protein tunnel suited for O2 diffusion to the heme.

Macrophage-generated oxygen- and nitrogen-reactive species control the development of Mycobacterium tuberculosis infection in the host. Mycobacterium tuberculosis 'truncated hemoglobin' N (trHbN) has been related to nitric oxide (NO) detoxification, in response to macrophage nitrosative stress, during the bacterium latent infection stage. The three-dimensional structure of oxygenated trHbN, solved at 1.9 A resolution, displays the two-over-two alpha-helical sandwich fold recently characterized in two homologous truncated hemoglobins, featuring an extra N-terminal alpha-helix and homodimeric assembly. In the absence of a polar distal E7 residue, the O2 heme ligand is stabilized by two hydrogen bonds to TyrB10(33). Strikingly, ligand diffusion to the heme in trHbN may occur via an apolar tunnel/cavity system extending for approximately 28 A through the protein matrix, connecting the heme distal cavity to two distinct protein surface sites. This unique structural feature appears to be conserved in several homologous truncated hemoglobins. It is proposed that in trHbN, heme Fe/O2 stereochemistry and the protein matrix tunnel may promote O2/NO chemistry in vivo, as a M.tuberculosis defense mechanism against macrophage nitrosative stress.

Amino Acid Sequence↗

PubMed2001

Very high resolution structure of a trematode hemoglobin displaying a TyrB10-TyrE7 heme distal residue pair and high oxygen affinity.

Monomeric hemoglobin from the trematode Paramphistomum epiclitum displays very high oxygen affinity (P(50)<0.001 mm Hg) and an unusual heme distal site containing tyrosyl residues at the B10 and E7 positions. The crystal structure of aquo-met P. epiclitum hemoglobin, solved at 1.17 A resolution via multiwavelength anomalous dispersion techniques (R-factor=0.121), shows that the heme distal site pocket residue TyrB10 is engaged in hydrogen bonding to the iron-bound ligand. By contrast, residue TyrE7 is unexpectedly locked next to the CD globin region, in a conformation unsuitable for heme-bound ligand stabilisation. Such structural organization of the E7 distal residue differs strikingly from that observed in the nematode Ascaris suum hemoglobin (bearing TyrB10 and GlnE7 residues), which also displays very high oxygen affinity. The oxygenation and carbonylation parameters of wild-type P. epiclitum Hb as well as of single- and double-site mutants, with residue substitutions at positions B10, E7 and E11, have been determined and are discussed here in the light of the protein atomic resolution crystal structure.

Amino Acid Sequence↗

PubMed2001

Single mutations at the subunit interface modulate copper reactivity in Photobacterium leiognathi Cu,Zn superoxide dismutase.

The functional properties and X-ray structures of five mutant forms of Photobacterium leiognathi Cu,Zn superoxide dismutase carrying single mutations at residues located at the dimer association interface have been investigated. When compared to the wild-type enzyme, the three-dimensional structures of the mutants show structural perturbations limited to the proximity of the mutation sites and substantial identity of active site geometry. Nonetheless, the catalytic rates of all mutants, measured at neutral pH and low ionic strength by pulse radiolysis, are higher than that of the wild-type protein. Such enzymatic activity increase is paralleled by enhanced active site accessibility to external chelating agents, which, in the mutated enzyme, remove more readily the active site copper ion. It is concluded that mutations at the prokaryotic Cu,Zn superoxide dismutase subunit interface can transduce dynamical perturbation to the active site region, promoting substrate active site accessibility. Such long-range intramolecular communication effects have not been extensively described before within the Cu,Zn superoxide dismutase homology family.

Amino Acid Substitution↗

PubMed2001

CD81 extracellular domain 3D structure: insight into the tetraspanin superfamily structural motifs.

Human CD81, a known receptor for hepatitis C virus envelope E2 glycoprotein, is a transmembrane protein belonging to the tetraspanin family. The crystal structure of human CD81 large extracellular domain is reported here at 1.6 A resolution. Each subunit within the homodimeric protein displays a mushroom-like structure, composed of five alpha-helices arranged in 'stalk' and 'head' subdomains. Residues known to be involved in virus binding can be mapped onto the head subdomain, providing a basis for the design of antiviral drugs and vaccines. Sequence analysis of 160 tetraspanins indicates that key structural features and the new protein fold observed in the CD81 large extracellular domain are conserved within the family. On these bases, it is proposed that tetraspanins may assemble at the cell surface into homo- and/or hetero-dimers through a conserved hydrophobic interface located in the stalk subdomain, while interacting with other liganding proteins, including hepatitis C virus E2, through the head subdomain. The topology of such interactions provides a rationale for the assembly of the so-called tetraspan-web.

Amino Acid Sequence↗

PubMed2001

An atomic force microscopy investigation of protein crystal surface topography.

Tapping mode atomic force microscopy was employed to study the surface structure of different protein crystals in a liquid environment. The (101) face of hen egg-white lysozyme crystals and the (111) face of horse spleen ferritin crystals were studied. On the (101) face of lysozyme crystals we observed islands delimitated by micro-steps and elongated in the [010] direction. The elongation direction coincides with the preferential growth direction predicted by a growth model reported in the literature. The islands observed on the ferritin (111) face are also delimitated by micro-steps but have circular symmetry. Sectioning of the images allowed us to measure the step heights. The surface free energy was estimated from the growth step morphology. Molecular resolution was achieved for ferritin crystals, showing a hexagonal surface packing, as expected for the molecular lattice of a (111) face in a fcc crystal.

Animals↗