Biomedical subjects

M Bolognesi

Publications and source records attributed to M Bolognesi.

At least 91 records · Page 5Linked to original sources

PubMed1997

Nutritional and prognostic significance of insulin-like growth factor 1 in patients with liver cirrhosis.

Most of the traditional parameters for nutrition assessment have important limitations in patients with chronic liver disease. Insulin-like growth factor 1 (IGF-1) has been found to be regulated by nutrition and proposed as a nutritional marker. Its nutritional significance in patients with liver cirrhosis, however, has not been investigated. Serum IGF-1 as well as traditional anthropometric, visceral, and immunologic parameters were evaluated in 64 hospitalized cirrhotics, followed up clinically for 2 y. IGF-1Z-score averaged -2.16 +/- 1.08 and inversely correlated with Child-Pugh score (P < 0.01), the most reliable composite score reflecting the severity of liver disease. IGF-1Z-score was not different in patients with or without signs of energy malnutrition, as defined by values of midarm muscle circumference (MAMC) and/or triceps skinfold (TSF) < 5th percentile. Moreover, IGF-1Z-score did not correlate with MAMC or TSF. Despite its correlation with all visceral proteins, the reduction of IGF-1 was much greater and more frequent than that of visceral proteins. Patients with IGF-1Z-score < median values (-2.5) showed lower long-term survival rates compared with patients with IGF-1Z-score > -2.5 (P < 0.01). These data indicate that serum IGF-1 is not related to energy malnutrition in cirrhotic patients, while it appears to be a good predictor of survival and an early marker of liver dysfunction. Multiple factors, most of which are related to the severity of the liver disease, may contribute to the reduction of IGF-1. This multifactorial pathogenesis probably accounts for its prognostic significance.

Adult↗

PubMed1997

Hemodynamic evaluation of the addition of isosorbide-5-mononitrate to nadolol in cirrhotic patients with insufficient response to the beta-blocker alone.

The association beta-blockers plus isosorbide-5-mononitrate (I5M) has been proposed for the treatment of portal hypertension in patients with insufficient response to beta-blockers alone, according to hemodynamic criteria. The mechanism of action in these patients is not clearly defined. Fifteen patients with cirrhosis and esophageal varices were evaluated by hepatic venous pressure gradient (HVPG) measurement and duplex-Doppler ultrasonography before and after 1 month of treatment with nadolol. Nine patients who did not exhibit a decrease in HVPG to 12 mm Hg or a percent decrease greater than 20% were classified as poor responders, and were studied again with the same methodology after 3 months of chronic administration of nadolol + I5M 20 mg twice per day. In poor responders, mean HVPG decrease after nadolol was 8.9% +/- 2.8%, and after the combination, it was 25.7% +/- 1.7% (P = .004). All patients except one became good responders to the association. Portal blood flow (PBF) decreased significantly after nadolol (P = .004), and remained unchanged after the addition of nitrates. Resistance to portal blood flow (RPBF) increased after nadolol (P = .02) and returned to baseline values during combined treatment (P = .03). In good responders, an adequate decrease in HVPG was associated with a decrease in PBF (P = .06) but no change in RPBF. A wide spectrum of combined changes in PBF and in RPBF after nadolol was observed in poor responders, ranging from no change in either parameter to a marked decrease in PBF counterbalanced by a marked increase in RPBF. The addition of I5M was followed in most cases by larger effects on resistance than on flow. Doppler parameters were not significantly correlated with the HVPG response to nadolol alone or associated with I5M. It is concluded that good hemodynamic responders to nadolol differ from poor responders in the lack of increase in RPBF after the drug. The addition of nitrates to nadolol is effective in decreasing portal pressure in most poor responders to nadolol alone. A decrease in outflow resistance is the main mechanism involved.

Adrenergic beta-Antagonists↗

PubMed1997

Dimensions of the intercondylar notch of the knee.

This study evaluates the three-dimensional contour of the intercondylar notch of the knee along its entire length. Forty-one distal femora, from human cadavers with a mean age of 45 years (range: 23 to 70 years) at the time of death were studied. The specimens were selected for the absence of previous surgery or degenerative changes including osteophyte formation. Measurements were taken from a cast of the distal femur. Transverse sections of the cast were cut every 3 mm in a plane perpendicular to the long axis of the femur. Within this plane, the epicondylar width, the condylar height, the notch height, and the minimum and maximum width of the notch were measured. The ratios between these measurements were calculated and subjected to analysis of variance. The notch width increased gradually from a mean of 1.8 cm at the distal aspect to a mean of 2.3 cm at the most proximal portion. The notch height increased from a mean value of 1.8 cm at the distal-most portion to a maximum of 2.4 cm at 1.8 cm from the distal-most portion of the notch. The height then decreased to a mean value of 1.3 cm at the most proximal portion of the notch. The epicondylar width:notch width ratio ranged from 4.70 distally to 4.25 proximally. These results indicate that the notch is narrowest distally and widens proximally, and the notch height is greatest in the midportion and decreases proximally and distally.

Adult↗

PubMed1996

Crystal structure of NH3-dependent NAD+ synthetase from Bacillus subtilis.

NAD+ synthetase catalyzes the last step in the biosynthesis of nicotinamide adenine dinucleotide. The three-dimensional structure of NH3-dependent NAD+ synthetase from Bacillus subtilis, in its free form and in complex with ATP, has been solved by X-ray crystallography (at 2.6 and 2.0 angstroms resolution, respectively) using a combination of multiple isomorphous replacement and density modification techniques. The enzyme consists of a tight homodimer with alpha/beta subunit topology. The catalytic site is located at the parallel beta-sheet topological switch point, where one AMP molecule, one pyrophosphate and one Mg2+ ion are observed. Residue Ser46, part of the neighboring 'P-loop', is hydrogen bonded to the pyrophosphate group, and may play a role in promoting the adenylation of deamido-NAD+ during the first step of the catalyzed reaction. The deamido-NAD+ binding site, located at the subunit interface, is occupied by one ATP molecule, pointing towards the catalytic center. A conserved structural fingerprint of the catalytic site, comprising Ser46, is very reminiscent of a related protein region observed in glutamine-dependent GMP synthetase, supporting the hypothesis that NAD+ synthetase belongs to the newly discovered family of 'N-type' ATP pyrophosphatases.

Amide Synthases↗

PubMed1996

N-ethoxycarbonyl-D-phenylalanyl-L-prolyl-alpha-azalysine p-nitrophenyl ester: a novel, high selective and optimal chromogenic active site titrant for human and bovine alpha-, beta- and gamma-thrombin.

The serine proteinase catalyzed hydrolysis of N-ethoxycarbonyl-D-phenylalanyl-L-prolyl-alpha-azalysine p- nitrophenyl ester (Eoc-D-Phe-Pro-azaLys-ONp) was investigated at pH 6.2 and 21.0 degrees C. The results are consistent with the minimum three-step catalytic mechanism. The acylation step is rate limiting for human (Lys 77 species) and porcine plasmin, and for bovine beta-trypsin, the deacylation rate being limiting, on the other hand, for human and bovine alpha-, beta- and gamma-thrombin. Moreover the M(r) 33,000 species of human urokinase and the neuraminidase-treated porcine pancreatic beta-kallikrein-B do not catalyze the hydrolysis of the tripeptide. According to the specificity properties of the serine proteinases considered. Eoc-D-Phe- Pro-azaLys-ONp shows the characteristics of a novel, high selective and optimal chromogenic active site titrant for human and bovine alpha-, beta- and gamma-thrombin.

Animals↗

PubMed1996

Crystal structure of D-amino acid oxidase: a case of active site mirror-image convergent evolution with flavocytochrome b2.

D-amino acid oxidase is the prototype of the FAD-dependent oxidases. It catalyses the oxidation of D-amino acids to the corresponding alpha-ketoacids. The reducing equivalents are transferred to molecular oxygen with production of hydrogen peroxide. We have solved the crystal structure of the complex of D-amino acid oxidase with benzoate, a competitive inhibitor of the substrate, by single isomorphous replacement and eightfold averaging. Each monomer is formed by two domains with an overall topology similar to that of p-hydroxybenzoate hydroxylase. The benzoate molecule lays parallel to the flavin ring and is held in position by a salt bridge with Arg-283. Analysis of the active site shows that no side chains are properly positioned to act as the postulated base required for the catalytic carboanion mechanism. On the contrary, the benzoate binding mode suggests a direct transfer of the substrate alpha-hydrogen to the flavin during the enzyme reductive half-reaction. The active site Of D-amino acid oxidase exhibits a striking similarity with that of flavocytochrome b2, a structurally unrelated FMN-dependent flavoenzyme. The active site groups (if these two enzymes are in fact superimposable once the mirror-image of the flavocytochrome b2 active site is generated with respect to the flavin plane. Therefore, the catalytic sites of D-amino acid oxidase and flavocytochrome b2 appear to have converged to a highly similar but enantiomeric architecture in order to catalvze similar reactions (oxidation of alpha-amino acids or alpha-hydroxy acids), although with opposite stereochemistry.

4-Hydroxybenzoate-3-Monooxygenase↗

PubMed1996

The allosteric regulation of pyruvate kinase.

Crystallographic and mutagenesis studies have unravelled the general features of the allosteric transition mechanism in pyruvate kinase. The enzyme displays a dramatic conformational change in going from the T- to the R-state. All three domains forming each subunit of the tetrameric enzyme undergo simultaneous and concerted rotations, in such a way that all subunit and domain interfaces are modified. This mechanism is unprecedented since in all tetrameric allosteric enzymes, characterised at atomic resolution, at least one of the domain or subunit interfaces remains unchanged on the T- to R-state transition. The molecular mechanism of allosteric regulation here proposed provides a rationale for the effect of single site mutations observed in the human erythrocyte pyruvate kinase associated with a congenital anaemia.

Allosteric Regulation↗

PubMed1996

Human alpha-thrombin inhibition by the active site titrant N alpha-(N,N-dimethylcarbamoyl)-alpha-azalysine p-nitrophenyl ester: a comparative kinetic and X-ray crystallographic study.

Kinetics for the hydrolysis of the chromogenic active site titrant N alpha-(N,N-dimethylcarbamoyl)-alpha-azalysine p-nitrophenyl ester (Dmc-azaLys-ONp) catalyzed by bovine beta-trypsin, bovine alpha-thrombin, human alpha-thrombin, human Lys77-plasmin, human urinary kallikrein, the M(r) 33,000 and M(r) 54,000 species of human urokinase, as well as by porcine pancreatic beta-kallikrein-A and B have been obtained between pH 6.0 and 8.0, at 21.0 degrees C. Moreover, the three dimensional structure of the human alpha-thrombin-(hirugen).Dmc-azaLys acyl.enzyme complex has been analyzed and refined by X-ray crystallography at 2.0 A resolution (R-factor = 0.168). As observed for bovine beta-trypsin, the acylating inhibitor molecule is covalently bound to the Ser195 catalytic residue, filling the human alpha-thrombin S1 primary specificity subsite with its lysyl side-group. However, the carbonyl group of the scissile human alpha-thrombin.Dmc-azaLys acyl bond does not occupy properly the oxyanion binding hole. At variance from the bovine beta-trypsin.Dmc-azaLys acyl.enzyme structure, a second, not covalently bound, inhibitor molecule, partly shielded by the 60-insertion loop of human alpha-thrombin, is contacting the enzyme "aryl-binding site".

Animals↗

PubMed1996

Structural bases for sulfide recognition in Lucina pectinata hemoglobin I.

The X-ray crystal structure of the sulfide derivative of ferric Lucina pectinata hemoglobin component I (HbI) has been determined at 1.9 A resolution (R-factor 0.186). The heme pocket structural organization of HbI is in keeping with its ligand binding properties. The fast sulfide association rate constant can be related to the presence of Gln(64)E7, as the heme distal residue, together with the protein structural properties in the CD-E distal region. Moreover, the very high sulfide affinity for HbI is reflected by the exceptionally slow ligand dissociation rate. The stabilization of the heme-bound sulfide molecule is achieved through hydrogen bonding to Gln(64)E7, as well as by finely tuned aromatic-electrostatic interactions with the clustered residues Phe(29)B10, Phe(43)CD1 and Phe(68)E11. Such a peculiar arrangement of phenylalanyl residues at the distal ligand binding site has not been observed before in the globin family, and is unique to HbI, a protein functionally devoted to sulfide transport.

Animals↗

PubMed1996

Three-dimensional structure of Xenopus laevis Cu,Zn superoxide dismutase b determined by X-ray crystallography at 1.5 A resolution.

Xenopus laevis Cu,Zn superoxide dismutase (recombinant isoenzyme b) has been crystallized and the structure determined at 1.49 A resolution. The crystals belong to space group P2(1)2(1)2(1), with cell constants a = 73.33, b = 68.86, c = 59.73 A, and contain one dimeric molecule of M(r) 32 000 per asymmetric unit. The structure was solved by molecular-replacement techniques using the semisynthetic Cu,Co bovine enzyme as search model, and refined by molecular dynamics with a crystallographic pseudo-energy term. During the final steps, positional and anisotropic thermal parameters of the atoms were refined. The R factor for the 49 209 unique reflections in the 10.0-1.49 A resolution range is 0.104, for a model comprising 2023 protein atoms, two Cu(2+), two Zn(2+), and 353 water molecules. The overall temperature factor for the model, including solvent, is 20.3 A(2), while the calculated r.m.s. coordinate error for the refined model is 0.036 A. As suggested by the primary structure homology to any other known intracellular eukaryotic superoxide dismutase (> 50%), the typical structural scaffolding of flattened antiparallel eight-stranded (beta-barrel is well conserved in X. laevis Cu,Zn superoxide dismutase b, together with the coordination geometry of the metal centers in the active site. The higher thermal stability of the bb X. laevis superoxide dismutase homodimer, with respect to dimers involving the a-type isoenzyme subunit(s), can be related, on the basis of the high-resolution structure, to side-chain and solvent interactions centered on residue Tyr149, in both b-type subunits. The analysis of the overall solvent structure reveals a number of equivalent water molecule sites in the two subunits, and in homologous superoxide dismutase models. Their locations are discussed in detail and classified on the basis of their structural role.

Journal Article↗

PubMed1996

Patterns in ionizable side chain interactions in protein structures.

In a selected set of 44 high-resolution, non-homologous protein structures, the intramolecular hydrogen bonds or salt bridges formed by ionizable amino acid side chains were identified and analyzed. The analysis was based on the investigation of several properties of the involved residues such as their solvent exposure, their belonging to a certain secondary structural element, and their position relative to the N- and C-termini of their respective structural element. It was observed that two-thirds of the interactions made by basic or acidic side chains are hydrogen bonds to polar uncharged groups. In particular, the majority (78%) of the hydrogen bonds between ionizable side chains and main chain polar groups (sch:mch bonds) involved at least one buried atom, and in 42% of the cases both interacting atoms were buried. In alpha-helices, the sch:mch bonds observed in the proximity of the C- and N-termini show a clear preference for acidic and basic side chains, respectively. This appears to be due to the partial charges of peptide group atoms at the termini of alpha-helices, which establish energetically favorable electrostatic interactions with side chain carrying opposite charge, at distances even greater than 4.5 angstrom. The sch:mch interactions involving ionizable side chains that belong either to beta-strands or to the central part of alpha-helices are based almost exclusively on basic residues. This results from the presence of main chain carbonyl oxygen atoms in the protein core which have unsatisfied hydrogen bonding capabilities.

Hydrogen Bonding↗

PubMed1996

Crystallization of NAD+ synthetase from Bacillus subtilis.

NAD(+)-synthetase is a ubiquitous enzyme catalyzing the last step in the biosynthesis of NAD+. Mutants of NAD+ synthetase with impaired cellular functions have been isolated, indicating a key role for this enzyme in cellular metabolism. Crystals of the enzyme from Bacillus subtilis suitable for x-ray crystallographic investigation have been grown from polyethylene glycol solutions. Investigation on the structural organization of NAD+ synthetase, an enzyme fundamental for NAD+ biosynthesis, and belonging to the recently characterized amidotransferase enzymatic family, will provide more insight into the catalytic mechanism of deamido-NAD+-->NAD+ conversion, a biosynthetic process that is a potential target for the development of antibiotic compounds against Bacillus sp. and related bacteria.

Amide Synthases↗

PubMed1996

Splenic Doppler impedance indices: influence of different portal hemodynamic conditions.

The spleen plays a pivotal role in the pathogenesis and maintenance of portal hypertension. Few data exist about splenic hemodynamics evaluated by duplex sonography in this condition. Twenty-six normal subjects, 207 patients with portal hypertension of various causes, and in different splenoportal hemodynamic conditions, and 31 patients with liver transplantation were evaluated. In each patient the splenic resistive index (RI = peak systolic--end diastolic velocity/peak systolic velocity) and pulsatility index (PI = peak systolic--end diastolic velocity/mean velocity) were measured. In 17 cirrhotic patients, splenic indices were compared with portal hemodynamics as invasively evaluated by hepatic vein catheterization. In the various groups, RI and PI were respectively: normal subjects, 0.51 +/- 0.05 and 0.72 +/- 0.11; cirrhotic patients with hepatopetal portal blood flow (n = 167), 0.64 +/- 0.08 and 1.03 +/- 0.24; cirrhotic patients with hepatofugal portal flow (n = 3), 0.74 +/- 0.08 and 1.27 +/- 0.08; cirrhotic patients with portal vein thrombosis (n = 9), 0.74 +/- 0.08 and 1.36 +/- 0.34; patients with noncirrhotic obstruction of the portal system (n = 7), 0.69 +/- 0.11 and 1.16 +/- 0.28; cirrhotic patients with surgical decompression of splenic vein system (n = 21), 0.54 +/- 0.07 and 0.76 +/- 0.15; patients with liver transplantation (n = 31), 0.50 +/- 0.08 and 0.70 +/- 0.15. Both RI and PI were significantly higher in cirrhotic patients with hepatopetal portal flow compared with controls (P < .0001), and even higher in cirrhotic patients with portal vein thrombosis (P < .004 and P < .001 in comparison with RI and PI values of cirrhotic patients). In patients with noncirrhotic portal vein thrombosis, splenic impedance indices were higher than those in controls (RI and PI P < .0001). Cirrhotic patients who underwent surgery for the therapy of portal hypertension showed splenic impedance indices significantly decreased compared with other cirrhotic patients (RI and PI P < .0001). In patients who underwent liver transplantation, splenic impedance indices were the same as those in controls. In 23 of the 52 patients surgically treated (surgical shunt or liver transplantation), impedance indices were evaluated both before and after surgical treatment. All these patients showed a decrease in splenic impedance indices (RI and PI, P < .0001) after surgical treatment. RI and PI values were higher in patients with large esophageal varices as compared with patients without or with small varices (P < .02 and P < .01). RI and PI values were not related to age, mean arterial pressure, sex, Child-Turcotte-Pugh score, presence of ascites, or cause. A significant correlation was found between splenic impedance indices and portal resistance as evaluated by hepatic vein catheterization (r = .80, P < .001 for RI values; r = .87, P < .001 for PI values). In conclusion, this study shows that splenic impedance indices are increased in cirrhotic patients, and seems to demonstrate that in patients with cirrhosis these indices reflect portal vein blood flow resistance.

Adult↗

PubMed1996

Crystallization and preliminary X-ray analysis of the monomeric Cu,Zn superoxide dismutase from Escherichia coli.

The Cu,Zn superoxide dismutase (Cu,Zn SOD) originally isolated from the periplasmic space of Escherichia coli has been cloned and overexpressed in the E. coli strain BMH 71/18. The protein has been purified as a single component of 17,000 Da, corresponding to one subunit of the common dimeric eukaryotic Cu,Zn SODs. Large crystals of the purified protein have been grown in the presence of polyethylene glycol 4,000 at pH 8.5; the crystals belong to the monoclinic space group P2(1), with unit cell constants a = 33.1 A, b = 52.6 A, c = 43.3 A, beta = 111.4 degrees. One SOD subunit is contained in the asymmetric unit, yielding a Vm value of 2.1 A3/Da; the crystals diffract X-rays beyond 2.0 A resolution.

Amino Acid Sequence↗

PubMed1996

Structural determinants of fluoride and formate binding to hemoglobin and myoglobin: crystallographic and 1H-NMR relaxometric study.

The x-ray crystal structure of the fluoride derivative of ferric sperm whale (Physeter catodon) myoglobin (Mb) has been determined at 2.5 A resolution (R = 0.187) by difference Fourier techniques. The fluoride anion, sitting in the central part of the heme distal site and coordinated to the heme iron, is hydrogen bonded to the distal His(64)E7 NE2 atom and to the W195 solvent water molecule. This water molecule also significantly interacts with the same HisE7 residue, which stabilizes the coordinated fluoride ion. Moreover, fluoride and formate binding to ferric Aplysia limacina Mb, sperm whale (Physeter catodon) Mb, horse (Caballus caballus) Mb, loggerhead sea turtle (Caretta caretta) Mb, and human hemoglobin has been investigated by 1H-NMR relaxometry. A strong solvent proton relaxation enhancement is observed for the fluoride derivatives of hemoproteins containing HisE7. Conversely, only a small outer-sphere contribution to the solvent relaxation rate has been observed for all of the formate derivatives considered and for the A. limacina Mb:fluoride derivative, where HisE7 is replaced by Val.

Animals↗

PubMed1996

New structures of allosteric proteins revealing remarkable conformational changes.

New three-dimensional structures of allosteric proteins reveal they have a flexible architecture that is instrumental to the regulation of protein function. Highlights are the structures of GroEL, pyruvate kinase, D-3-phosphoglycerate dehydrogenase and the acetylcholine receptor. Furthermore, significant progress in understanding the nature of the intermediates involved in an allosteric reaction has been achieved through recent spectroscopic and crystallographic studies on haemoglobin.

Allosteric Regulation↗

PubMed1996

Malnutrition in alcoholic and virus-related cirrhosis.

The study aimed to define the prevalence, characteristics, and clinical importance of nutritional disorders in patients with liver cirrhosis. Nutritional status was evaluated in 120 hospitalized patients--77 with alcoholic and 43 with virus-related cirrhosis--by anthropometric, visceral, and immunologic measurements. Energy malnutrition, defined as triceps skinfold thickness (TSF) and/or midarm muscle circumference (MAMC) below the 5th percentile of standard values, was found in 34% of the study population. Patients below the 5th percentile for MAMC and/or TSF showed significantly lower survival rates at e, 6, 12, and 24 mo compared with patients above the 5th percentile. Protein malnutrition (low albumin, transthyretin, transferrin, and retinol-binding-protein concentrations) and immunoincompetence (abnormal response to skin tests) were much more frequent (81% and 59%) than energy malnutrition (34%). Serum proteins correlated with the degree of liver function impairment, but not with immunologic tests. The prevalence, characteristics, and severity of protein-energy malnutrition were comparable in alcoholic and viral cirrhosis. Malnutrition was correlated with the clinical severity of the liver disease. The study shows that protein-energy malnutrition is a common complication of liver cirrhosis. Nutritional disorders appear to be related to the degree of liver injury rather than to its etiology. Compared with other methods, which have important limitations in liver disease, anthropometry is currently the most reliable method for nutritional assessment in clinical practice and may be valuable for predicting survival in cirrhotic patients.

Adult↗

PubMed1996

Optimizing the time-frame for the definition of bleeding-related death after acute variceal bleeding in cirrhosis.

OBJECTIVE: To identify the best time-frame for defining bleeding-related death after variceal bleeding in patients with cirrhosis. DESIGN: Prospective long-term evaluation of a cohort of 155 patients admitted with variceal bleeding. SETTING: Eight medical departments in seven hospitals in north-eastern Italy. METHODS: Non-linear regression analysis of a hazard curve for death, and Cox's multiple regression analyses using different zero-time points. RESULTS: Cumulative hazard plots gave two slopes, the first corresponding to the risk of death from acute bleeding, the second a baseline risk of death. The first 30 days were outside the confidence limits of the regression curve for the baseline risk of death. Using Cox's regression analysis, the significant predictors of overall mortality risk were balanced between factors related to severity of bleeding and those related to severity of liver disease. If only deaths occurring after 30 days were considered, only predictors related to the severity of liver disease were found to be of importance. CONCLUSION: Thirty days after bleeding is considered to be a reasonable time-frame for the definition of bleeding-related death in patients with cirrhosis and variceal bleeding.

Adult↗