Acute abdominal aortic thrombosis caused by paroxysmal atrial fibrillation.

Acute abdominal aortic thrombosis is a rare and potential fatal event, which occurs in adult subjects. We present the case of a 72-year-old-man, who referred to the emergency Department of our hospital because of persistent severe abdominal and perineal pain. Doppler ultrasounds and computerized tomography angiography revealed the acute thrombosis of the abdominal aorta. Immediate revascularization through aortic thrombo-endoarterectomy resolved the disease.

Differential role of EGF and BFGF in human GBM-TIC proliferation: relationship to EGFR-tyrosine kinase inhibitor sensibility.

Glioblastoma multiforme (GBM) is among the most devastating human tumors being rapidly fatal despite aggressive surgery, radiation and chemotherapies. It is characterized by extensive dissemination of tumor cells within the brain that hinders complete surgical resection. GBM tumor initiating-cells (TICs) are a rare subpopulation of cells responsible for tumor development, growth, invasiveness and recurrence after chemotherapy. TICs from human GBM can be selected in vitro using the same conditions permissive for the growth of normal neural cells, of which share some features including marker expression, self-renewal capacity, long-term proliferation, and ability to differentiate into neuronal and glial cells. EGFR overexpression and its constitutive activation is one of the most important signaling alteration identified in GBM, and its pharmacological targeting represents an attractive therapeutic goal. We previously demonstrated that human GBM TICs have different sensitivity to the EGFR kinase inhibitors erlotinib and gefitinib, depending on the differential modulation of downstream signaling cascades. In this work we investigated the mechanisms of resistance to erlotinib in two human GBM TIC cultures, analyzing EGF and bFGF individual contribution to proliferation, clonogenicity, and migration. We demonstrated the presence of a small cell subpopulation whose proliferation is supported by EGF and a larger one mainly dependent on bFGF. Thus, insensitivity to EGFR kinase inhibitors as far as TIC proliferation results from a predominant FGFR activation that hides the inhibitory effects induced on EGFR signaling. Conversely, EGF and bFGF induced cell migration with similar efficacy. In addition, unlike neural stem/progenitors cells, the removal of chondroitin sulphate proteoglycans from cell surface was unable to discern EGF- and bFGF-dependent subpopulations in GBM TICs.

ADMA, SDMA, L-Arginine and nitric oxide in allergic pediatric bronchial asthma.

Published data regarding asymmetric dimethylarginine (ADMA), symmetric dimethylarginine (SDMA), L-arginine (L-ARG) and nitric oxide fraction in exhaled air (FeNO) in pediatric bronchial asthma are limited. Many question remain open about plasma concentration of these substances. The aim of this study is to evaluate ADMA, SDMA, L-ARG and FeNO concentration in allergic pediatric mild asthmatic patients in respect to healthy subjects. In this case-control study 60 children (50 asthmatics and 10 healthy) underwent a complete clinical visit, baseline respiratory function, allergy tests and biochemical analyses. The statistical significance of the different concentrations between the two groups were studied using one-way analysis of variance (ANOVA). A p value less than 0.05 was considered statistically significant. The mean plasma ADMA (0.58 vs 0.68 micromol/L), SDMA (0.40 vs 0.45 micromol/L) and L-ARG (52.2 vs 74.13 micromol/L) concentration were significantly lower (p less than 0.001) in the asthmatic patients in respect to healthy subjects (control group). The concentration of FeNO was significantly higher in the asthmatic subjects in respect to the control group (9.18 vs 4.2 micromol/L; p less than 0.001). Low plasma concentrations of ADMA, SDMA, L-ARG and high concentration of FeNO are associated with bronchial asthma and indicate an important role in airway disease through NO metabolism.

Lycopene and preclinical carotid atherosclerosis.

Evidence from epidemiological and clinical studies suggests a possible correlation between serum antioxidant levels and cardiovascular disease risk. High plasma concentrations of lycopene have been associated with reduced prevalence of cardiovascular disease. The aim of this study is to compare plasma concentrations of lycopene in subjects with or without ultrasonic evidence of asymptomatic carotid atherosclerosis. One hundred and twenty subjects underwent physical examination, ultrasonic measurement of common carotid artery intima-media thickness and serum profile analysis. Logistic regression methods and analysis of variance were used to determine whether differences existed between participants with or without evidence of carotid atherosclerosis. Of the 120 participants, 58 exhibited evidence of carotid atherosclerosis. Participants with ultrasonic evidence of carotid atherosclerosis exhibited significantly higher serum concentrations of total cholesterol, LDL-cholesterol and triglycerides. In contrast, participants with ultrasonic evidence of carotid atherosclerosis exhibited significantly lower plasma concentrations of lycopene. These data suggest that higher serum levels of lycopene may play a protective role versus cardiovascular diseases, in particular carotid atherosclerosis.

Recurrent atrial fibrillation in a patient with ulcerative colitis treated with azathioprine: case report and review of the literature.

We present a clinical case report regarding recurrent atrial fibrillation in a patient with ulcerative colitis treated with azathioprine. Atrial fibrillation represents the most common sustained cardiac arrhythmia, occurring in 1-2% of the general population and characterized by seemingly disorganized atrial depolarizations without effective atrial contraction. Several mechanisms determine this arrhythmia; in particular remodelling (structural, mechanical and electrical alteration related to atrial fibrillation). The pro-arrhythmic effect of azathioprine may be evaluated during immunosuppressive therapy to be aware of this serious but reversible adverse effect.

Relationship between asymmetric dimethylarginine and asymptomatic carotid atherosclerosis.

Asymmetric dimethylarginine (ADMA) is an endogenous inhibitor of nitric oxide (NO) associated with an increased risk of cardiovascular disease (CVD). In this study we assessed the relationship between ADMA and asymptomatic carotid intima-media thickness (CIMT). Eighty subjects underwent a complete history and physical examination, determination of serum chemistries and ADMA levels, and carotid ultrasound investigation (CUI). None of the subjects had symptoms of carotid atherosclerosis and nor were they taking any medication. Statistical analyses showed that high plasma levels of ADMA were positively correlated to CIMT (p less than 0.001). Total cholesterol, low density lipoprotein cholesterol, triglycerides and C-reactive protein plasma concentrations were significantly associated with asymptomatic carotid atherosclerosis (p less than 0.001). High serum concentrations of ADMA were associated with early carotid atherosclerotic lesions as measured by CIMT and represent a new marker of asymptomatic carotid atherosclerosis.

The interaction of humic substances with the human prion protein fragment 90-231 affects its protease K resistance and cell internalization.

In this paper we analyzed the determinants and the structural effects of the interaction of human prion protein fragment 90-231 (HuPrP) with humic substances, (HS) including humic (HA) and fulvic (FA) acids, natural refractory organic polyanions widely diffused in soils and waters. We show that this interaction is mainly driven by non-specific electrostatic attraction involving regions situated within alpha-helix A and beta-sheet S1 of human PrP. FA binding to HuPrP altered its ability to acquire some PrPSc-like characteristics induced by the mild thermal denaturation of the peptide (1 h at 53 degrees C). In particular, in the presence of FA, HuPrP shows a reduced amount of beta-sheet content (as demonstrated by the reduced binding of thioflavin T), an increased sensitivity to protease K and an inhibition of the entering in the fibrillogenic pathway. FA/HuPrP interaction caused the aggregation of the peptide in unstructured macrocomplexes, as demonstrated by the altered electrophoretic migration in semi-denaturing detergent-agarose gel assay. Importantly, in the presence of FA the rate of internalization of HuPrP in human neuroblastoma cells was significantly reduced as compared to that of the beta-structured peptide. Therefore, HS inhibited the acquisition of PrP(Sc)-like structural properties that, in turn, are responsible for HuPrP intracellular accumulation and lead to neuronal death. Important implications of these data are that HuPrP-HS complexes, being unable to be internalized in living cells may represent a molecular mechanism for the reduced transmission of prion transmission from HS-rich soil also in the presence of contamination from infected animals.

[Surveillance groups and occupational phisicians for prevention and security in work environment: legal aspects]. / Organi di vigilanza e medici competenti per la prevenzione e la sicurezza nei luoghi di lavoro: aspetti giuridici.

Italian jurisdiction, since the setting up of the Republic in 1948, has experienced a real and proper Copernican revolution, not only in the field of prevention of accidents and safety at work, which has meant that at the centre of the system there is no longer a State-finality but the individual person, in the form of inviolable rights that the Democratic Republic-vehicle recognises and guarantees in every place, including all places of occupation. To this end the entrepreneur/employer has become the guarantor of the health of his employees through a complex series of laws and regulations that imposes the adoption of safety measures on an individual, general and organizational level in the company. This guarantee has been reinforced by bringing in penalties for all those evasions or violations of these obligations of the entrepreneur/employer. The centralizing of the culture of prevention has meant that all the controls converge on the judicial police, with the head of the surveillance bodies having the status of officer of the judicial police, and with the adoption of regulations that follow the code of criminal procedures, which, while being binding for the same surveillance bodies is also a guarantee in respecting the right to be defended of the entrepreneur/employer.

Conformation dependent pro-apoptotic activity of the recombinant human prion protein fragment 90-231.

The transition of prion protein from a mainly alpha-structured isoform (PrPC) to a beta sheet-containing protein (PrPSc) represents a major pathogenetic mechanism in prion diseases. To study the role of PrP structural conformation in prion-dependent neurodegeneration, we analysed the neurotoxicity of PrP in alpha and beta conformations, using a recombinant protein encompassing amino acids 90-231 of the human PrP (hPrP90-231). Using controlled thermal denaturation (53 degrees C, 1h) we converted hPrP90-231 in a structural isoform displaying PrPSc-related characteristics: high beta sheet content, increased aggregability and a slight increase in the resistance to protease K. In virtue of these structural changes, hPrP90-231 powerfully affected the survival of SH-SY5Y cells, inducing a caspase-3 and p38- dependent apoptosis. Conversely, in the native alpha-helix-rich conformation, hPrP90-231 did not show significant cell toxicity. The relationship between the structural state of hPrP90-231 and its neurotoxicity was demonstrated, inducing the thermal denaturation of the peptide in the presence of Congo red that prevented both the transition of hPrP90-231 into a beta-rich isoform and the acquisition of toxic properties. In conclusion, we report that the toxicity of hPrP90-231 is dependent on its three-dimensional structure, as is supposed to occur for the pathogen PrP during TSE.

Identification of a conserved N-capping box important for the structural autonomy of the prion alpha 3-helix: the disease associated D202N mutation destabilizes the helical conformation.

Peptides corresponding to three alpha helices present in the C-terminal region of the human prion protein have been synthesized and their structural autonomy analyzed by circular dichroism (CD) and NMR spectroscopy. The results obtained indicate that the protein fragment corresponding to the alpha 3-helix, in contrast to alpha 1 and alpha 2 peptides, shows a complete structural autonomy. The chemical shifts values found for NH and CHalpha resonance of the isolated alpha 3 peptide, formed by 30 aminoacid residues, were markedly and surprisingly similar to the corresponding values of the alpha 3-helix in the protein. The structural autonomy of the alpha 3-helix is profoundly determined by the presence of the conserved capping box and, in part, by the ionic bond formed between Glu200 and Lys204. On the basis of these observations a novel PrP consensus pattern, centered on the alpha 3-helix region, has been defined. The data indicate that this autonomous and highly conserved region of the PrPc likely plays a critical role in folding and stability. This gives an explanation of why many of pathogenic mutations occur in this part of the molecule, sharing relevant effects on the overall protein conformation. In particular the D202N capping mutation almost completely destabilizes the isolated alpha 3 peptide. While it is well known that the D202N substitution is associated with a GSS disease, the possible structural basis of this fatal pathology has never been investigated. We propose that a lower alpha 3-helical propensity leading to a major destabilization of the PrPc molecule initiates the pathogenic process associated with D202N capping mutation.

Purification and characterisation of two GST's forms from Rhizobium leguminosarum with a high affinity to herbicides.

Cytosolic glutathione transferases are a family of multifunctional proteins that catalyse the conjugation of GSH to a large variety of endogenous and exogenous compounds. These enzymes have been widely studied in mammals and, to a lesser extent, in plants. In plants, GSTs can detoxify herbicides; they are also induced by pathogenic infection and are likely to be involved in defence responses. GSTs are found in pathogenic and not pathogenic prokaryotes but the functional role played by these enzymes in the cell still remains to be clarified. Here we report the purification and characterisation of two GST forms from Rhizobium leguminosarum that play a very important role in agriculture by inducing nitrogen-fixing nodules on the roots of legumes. These bacterial GSTs from R. leguminosarum have immunological characteristics that are different among them and they are characterised both by a high affinity to herbicides.

Prion protein fragment 106-126 induces a p38 MAP kinase-dependent apoptosis in SH-SY5Y neuroblastoma cells independently from the amyloid fibril formation.

Prion diseases are neurodegenerative disorders of the central nervous system of humans and animals, characterized by spongiform degeneration of the central nervous system, astrogliosis, and deposition of amyloid into the brain. The conversion of a cellular glycoprotein (prion protein, PrP(C)) into an altered isoform (PrP(Sc)) has been proposed to represent the causative event responsible for these diseases. The peptide corresponding to the residues 106-126 of PrP sequence (PrP106-126) is largely used to explore the neurotoxic mechanisms underlying the prion diseases. We investigated the intracellular signaling responsible for PrP106-126-dependent cell death in the SH-SY5Y human neuroblastoma cell line. In these cells, PrP106-126 treatment induced apoptotic cell death and the activation of caspase-3. The p38 MAP-kinase blockers (SB203580 and PD169316) prevented the apoptotic cell death evoked by PrP106-126 and Western blot analysis revealed that the exposure of the cells to the peptide induced p38 activation. However, whether the neuronal toxicity of PrP106-126 is caused by a soluble or fibrillar form of this peptide is still unknown. In this study, we correlated the structural state of this peptide with its neurotoxicity. We show that the two conserved glycines in position 114 and 119 prevent the peptide to assume a structured conformation, favoring its aggregation in amyloid fibrils. The substitution of both glycines with alanine residues (PrP106-126AA) generates a soluble nonamyloidogenic peptide, that retained its toxic properties when incubated with neuroblastoma cells. These data show that the amyloid aggregation is not necessary for the induction of the toxic effects of PrP106-126.

Expression in E. coli and purification of recombinant fragments of wild type and mutant human prion protein.

Prion diseases are fatal neurodegenerative disorders of the CNS of men and animals, characterized by spongiform degeneration of the CNS, astrogliosis and deposition of amyloid into the brain. The conversion of a cellular glycoprotein (the prion protein, PrP(C)) into an altered isoform (the prion scrapie, PrP(Sc)), which accumulates within the brain tissue by virtue of its resistance to the intracellular catabolism, is currently believed to represent the etiologic agent responsible for these diseases. Synthetic or recombinant polypeptides are commonly used to elucidate the mechanism of proteins involved in neurodegenerative diseases. Here we describe a procedure, which allows the synthesis and purification in its native folding, of the human prion protein fragment 90-231, corresponding to the protease resistant core of PrP(Sc). We synthesized the polypeptides 90-231 of both the wild type and the E200K mutant isoforms of PrP. Using a gluthatione S-transferase (GST) fusion protein approach, milligram amounts of polypeptides were obtained after expression in E. coli. The recovery of the purified fusion protein was monitored following the evaluation of the GST activity. The PrP fragment was released from the fusion protein immobilized on a glutathione-coupled agarose resin by direct cleavage with thrombin. The recombinant protein was identified by comassie stained acrylamide gel and by immunoblotting employing a monoclonal anti-PrP antibody. The peptide purified by gel filtration chromatography showed mainly an alpha-helix structure, as analysed by circular dichroism (CD) and an intact disulfide bridge. The same procedure was also successfully employed to synthesize and purify the E200K mutant PrP fragment.

The folding and stability of human alpha class glutathione transferase A1-1 depend on distinct roles of a conserved N-capping box and hydrophobic staple motif.

An N-capping box and a hydrophobic staple motif are strictly conserved in the core of all known glutathione S-transferases (GST). In the present work, mutations of hGSTA1-1 enzyme residues forming these motifs have been generated. The analysis of S154A, D157A, and S154A/D157A capping mutants indicate that the removal of this local signal destabilizes the protein. The fact that the third helical residue D157A mutation (N-3) was much more destabilizing than the first helical residue S154A mutation (N-cap) suggests that the appropriate conformation of the conserved substructure formed by the alpha 6-helix and preceding loop (GST motif II) is crucial for the overall protein stability. The refolding study of GSTA1-1 variants supports the prediction that this subdomain could represent a nucleation site of refolding. The analysis of L153A, I158A, L153G, and L153A/I158A hydrophobic staple mutants indicate that the removal of this motif destabilizes the GSTA1-1 structure as well as its refolding transition state. The hydrophobic staple interaction favors essential inter-domain contacts and, thereby, in contrast to capping interactions, accelerates the enzyme reactivation. Its strict conservation in the GST system supports the suggestion that this local signal could represent an evolutionarily conserved determinant for rapid folding.

Conformational properties of five peptides corresponding to the entire sequence of glutathione transferase domain II.

Five peptides matching the helices alpha4, alpha5, alpha6, alpha7, and alpha8, spanning the entire sequence of domain II of pG-STP1-1, have been synthesized and their conformations analyzed by far-UV CD spectroscopy. The results show that a5, a7, and a8 peptides are unstructured in water/2,2,2-trifluoroethanol (TFE) solutions. The a4-peptide also adopts random conformations in aqueous solvent. Moreover, the relative low helical content (20%), estimated for this peptide in the presence of 30% (v/v) TFE, suggests that the sequence of this protein fragment does not possess sufficient information for a strong helical propensity. On the contrary, the synthesized a6 peptide, in the presence of TFE, showed a relevant structural autonomy with a helical content (41%) which was significantly higher than that estimated, under the same conditions, for all other peptides. More in general in the presence of solvents less polar than water, the isolated a6 peptide shows the same helical conformation adopted by the corresponding alpha6-helix in the hydrophobic core of the protein. A n-capping box motif, strictly conserved at the N-terminal of the alpha6-helix of all GST and related protein including eucaryotic translation elongation factor (EF1gamma) and the yeast prion protein Ure2, plays an important role in the alpha-helix nucleation and stability of this protein fragment. The results suggest that the alpha6-helix might represent a nucleation site of GST folding and that the helical conformation of this region of the protein is an important requirement during earlier events of GST refolding.

Structures of thermolabile mutants of human glutathione transferase P1-1.

An N-capping box motif (Ser/Thr-Xaa-Xaa-Asp) is strictly conserved at the beginning of helix alpha6 in the core of virtually all glutathione transferases (GST) and GST-related proteins. It has been demonstrated that this local motif is important in determining the alpha-helical propensity of the isolated alpha6-peptide and plays a crucial role in the folding and stability of GSTs. Its removal by site-directed mutagenesis generated temperature-sensitive folding mutants unable to refold at physiological temperature (37 degrees C). In the present work, variants of human GSTP1-1 (S150A and D153A), in which the capping residues have been substituted by alanine, have been generated and purified for structural analysis. Thus, for the first time, temperature-sensitive folding mutants of an enzyme, expressed at a permissive temperature, have been crystallized and their three-dimensional structures determined by X-ray crystallography. The crystal structures of human pi class GST temperature-sensitive mutants provide a basis for understanding the structural origin of the dramatic effects observed on the overall stability of the enzyme at higher temperatures upon single substitution of a capping residue.

Validation of a new technique to assess exhaled hydrogen peroxide: results from normals and COPD patients.

Chronic airways inflammation in chronic obstructive pulmonary disease (COPD) induces the activation of several cell types with delivery of proteases and reactive oxygen species (ROS). Assessing oxidant content in the exhaled air of COPD patients has proven useful in monitoring airway inflammation. The present study was designed to confirm the usefulness of exhaled hydrogen peroxide concentration determination in COPD patients using a new technique which allows longer storage of the expired air condensate before the H2O2 assay. The technique was applied in 13 healthy nonsmoking subjects (six male, age range 22-40 yrs) and in seven patients (five male, age range 58-81 yrs) with mild or moderate COPD. Subjects breathed into a one-valve mouthpiece, and the exhaled air was directed into a vial kept at 0 degree C. After approximately 15 min of quiet breathing, 1 mL of expired air condensate was collected. An aliquot, 450 microL, of this sample was immediately added to an equal volume of a reaction mixture containing 2 mM 3,5,3',5'-tetramethylbenzidine and 40 microL of enzyme stock solution (0.5 mg.mL-1). After 15 min, 45 microL sulphuric acid was added (1 N final concentration), resulting in a reaction mixture pH of 1.0. After a further 10-min incubation, H2O2 concentration determination was performed spectrophotometrically at 450 nm. This solution, as well as the H2O2 assay, was stable for > or = 24 h if the sample was kept in the dark and at 4 degrees C. There was high stability on repeated measures, with a coefficient of variation equal to zero. The mean +/- SD H2O2 level in exhaled air from normal subjects was 0.12 +/- 0.09 microM, whereas it was significantly increased in COPD patients (0.50 +/- 0.11 microM; p = 0.0001 compared to healthy subjects). In three healthy control subjects, a normal H2O2 level in expired air increased to 0.70-0.80 microM during an acute upper respiratory tract infection. This new technique of hydrogen peroxide assay in expired air condensate greatly minimizes the inaccuracy deriving from the instability of hydrogen peroxide. The preliminary results obtained using this technique provide direct evidence for increased reactive oxygen species production in the airways of stable chronic obstructive pulmonary disease patients. However, the specificity of the procedure could be reduced by the interference of upper respiratory tract infections.

Structural characterization of acid-induced intermediates of human glutathione transferase P1-1.

The acid denaturation of human glutathione transferase P1-1 (hGSTP1-1) has been performed to investigate the unfolding intermediates of the protein and their possible involvement in the refolding mechanism. The acid-induced structures of GSTP1-1 have been characterized by activity, gel filtration, intrinsic fluorescence and far-u.v. circular dichroism (CD) techniques. Because of the non-identity of the different transitions monitored, the acid denaturation of hGSTP1-1 appears to be a multistep process during which several intermediates coexist in equilibrium. The dependence of inactivation on the protein concentration, as well as gel-filtration experiments, indicate that the inactivation transition, centred at about pH 4.0, corresponds to the monomerization of the protein. At pH 2.0, when the enzyme is completely inactive, the protein retains a small, but significant, amount of secondary structure. This means that the dimeric arrangement of the molecule is important for maintaining the native-like secondary structure of the monomer. The results show that, at low pH, the compact state of the GST monomer, even upon the addition of salts, does not possess native-like secondary structure as described for many monomeric proteins (molten globule). In the presence of physiological concentrations of salts, the protein solution at pH 2.0 leads to a dead-end aggregation process, suggesting that this compact state cannot represent a productive intermediate of the refolding pathway.

A conserved "hydrophobic staple motif" plays a crucial role in the refolding of human glutathione transferase P1-1.

The specific (i, i+5) hydrophobic staple interaction involving a helix residue and a second residue located in the turn preceding the helix is a recurrent motif at the N terminus of alpha-helices. This motif is strictly conserved in the core of all soluble glutathione transferases (GSTs) as well as in other protein structures. Human GSTP1-1 variants mutated in amino acid Ile(149) and Tyr(154) of the hydrophobic staple motif of the alpha6-helix were analyzed. In particular, a double mutant cycle analysis has been performed to evaluate the role of the hydrophobic staple motif in the refolding process. The results show that this local interaction, by restricting the number of conformations of the alpha6-helix relative to the alpha1-helix, favors the formation of essential interdomain interactions and thereby accelerates the folding process. Thus, for the first time it is shown that the hydrophobic staple interaction has a role in the folding process of an intact protein. In P(i) class GSTs, Tyr(154) appears to be of particular structural importance, since it interacts with conserved residues Leu(21), Asp(24), and Gln(25) of the adjacent alpha1-helix which contributes to the active site. Human GSTP1-1 variants L21A and Y154F have also been analyzed in order to distinguish the role of interdomain interactions from that of the hydrophobic staple. The experimental results reported here suggest that the strict conservation of the hydrophobic staple motif reflects an evolutionary pressure for proteins to fold rapidly.