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1.
Oncogene ; 28(20): 2074-86, 2009 May 21.
Article in English | MEDLINE | ID: mdl-19377510

ABSTRACT

Resistance to detachment-induced apoptosis, a process commonly referred as anoikis, is emerging as a hallmark of metastatic malignancies, mainly because it can ensure anchorage-independent growth and survival during organ colonization. Besides, a sustained oxidative stress has been associated with several steps of carcinogenesis, including transformation and achievement of a motile mesenchymal phenotype. Here, we demonstrate that metastatic prostate carcinoma cells, undergoing a constitutive deregulated production of reactive oxygen species due to sustained activation of 5-lipoxygenase, lack suicidal pathways in response to lack of matrix contact. These amplified and persistent redox signals in PC3 cells leads to maintenance of Src oxidation and activation in the absence of adhesion, thereby sustaining a ligand-independent phosphorylation of epidermal growth factor receptor. This leads to chronic activation of pro-survival signals, culminating in degradation of the pro-apoptotic protein Bim, thereby promoting cell survival even in the absence of proper adhesion. Anoikis sensitivity of metastatic cells is restored with antioxidant intervention or genetic manipulation of the redox-mediated pro-survival pathway, as well as exposure to a pro-oxidant environment strongly increases anoikis resistance in non-transformed prostate epithelial cells. Hence, our results allow new insight into the aetiology of the molecular mechanisms granting anoikis resistance of metastatic cancers, opening new avenues to pharmacological intervention for antioxidant-sensitive invasive tumours.


Subject(s)
Anoikis/physiology , ErbB Receptors/metabolism , Prostatic Neoplasms/metabolism , Prostatic Neoplasms/pathology , Proto-Oncogene Proteins pp60(c-src)/metabolism , Reactive Oxygen Species/metabolism , Antioxidants/pharmacology , Apoptosis/physiology , Apoptosis Regulatory Proteins/metabolism , Arachidonate 5-Lipoxygenase/genetics , Arachidonate 5-Lipoxygenase/metabolism , Bcl-2-Like Protein 11 , Blotting, Western , Cells, Cultured , Epithelial Cells/metabolism , ErbB Receptors/genetics , Flow Cytometry , Humans , Immunoprecipitation , Lipoxygenase Inhibitors , Male , Membrane Proteins/metabolism , Oxidants/pharmacology , Oxidation-Reduction , Oxidative Stress , Phosphorylation , Prostate/metabolism , Prostate/pathology , Prostatic Neoplasms/secondary , Proto-Oncogene Proteins/metabolism , Proto-Oncogene Proteins pp60(c-src)/genetics
2.
Cell Death Differ ; 15(5): 867-78, 2008 May.
Article in English | MEDLINE | ID: mdl-18259192

ABSTRACT

Proper attachment to the extracellular matrix (ECM) is essential for cell survival. The loss of integrin-mediated cell-ECM contact results in an apoptotic process termed anoikis. However, mechanisms involved in regulation of cell survival are poorly understood and mediators responsible for anoikis have not been well characterized. Here, we demonstrate that reactive oxygen species (ROS) produced through the involvement of the small GTPase Rac-1 upon integrin engagement exert a mandatory role in transducing a pro-survival signal that ensures that cells escape from anoikis. In particular, we show that ROS are responsible for the redox-mediated activation of Src that trans-phosphorylates epidermal growth factor receptor (EGFR) in a ligand-independent manner. The redox-dependent phosphorylation of EGFR activates both extracellular signal-regulated protein kinase and Akt downstream signalling pathways, culminating in degradation of the pro-apoptotic protein Bim. Hence, our results shed new light on the mechanism granting the adhesion-dependent antiapoptotic effect, highlighting a fundamental role of ROS-mediated Src regulation in ensuring anoikis protection.


Subject(s)
Anoikis/physiology , Cell Survival/physiology , Oxidation-Reduction , Reactive Oxygen Species/metabolism , Apoptosis Regulatory Proteins/metabolism , Bcl-2-Like Protein 11 , Cell Adhesion/physiology , Cell Line , Enzyme Activation , ErbB Receptors/metabolism , Extracellular Matrix/metabolism , Extracellular Signal-Regulated MAP Kinases/metabolism , Humans , Integrins/metabolism , Membrane Proteins/metabolism , Proto-Oncogene Proteins/metabolism , Proto-Oncogene Proteins c-akt/metabolism , Signal Transduction/physiology , Transcriptional Activation , rac1 GTP-Binding Protein/metabolism , src-Family Kinases/metabolism
3.
Recenti Prog Med ; 97(2): 74-8, 2006 Feb.
Article in Italian | MEDLINE | ID: mdl-16671271

ABSTRACT

Retrospective research carried out by 29 General Practitioners in their databases, in order to evaluate the prevalence of gastro-esophageal reflux disease in its different clinical outbreaks and the incidence of new diagnosis in the last quinquennium, the diagnostic approach through instrumental examinations (endoscopy) or empirical tests (PPI test), and the therapeutical aspects, in particular concerning the usage of PPI. The prevalence has been of 3.82%, while the data concerning the incidence have pointed out a progressive increase of the diagnosis in the last quinquennium, specially for the atypical outbreaks. Moreover, it has been noted a likely excessive use of endoscopy, in the follow up as well, while less used is the IPP test. Gastroesophageal reflux disease is the most important item in the expenditure for the usage of IPP.


Subject(s)
Gastroesophageal Reflux , Primary Health Care/statistics & numerical data , Adult , Aged , Diagnosis, Differential , Esophagoscopy , Female , Gastroesophageal Reflux/diagnosis , Gastroesophageal Reflux/epidemiology , Gastroesophageal Reflux/therapy , Gastrointestinal Agents/therapeutic use , Gastroscopy , Humans , Hydrogen-Ion Concentration , Incidence , Italy/epidemiology , Male , Middle Aged , Prevalence , Proton Pump Inhibitors
4.
Cell Mol Life Sci ; 62(9): 931-6, 2005 May.
Article in English | MEDLINE | ID: mdl-15798896

ABSTRACT

Protein tyrosine phosphatases (PTPs) have been generally recognised as key modulators of cell proliferation, differentiation, adhesion and motility. During signalling, several PTPs undergo two posttranslational modifications that greatly affect their enzymatic activity: tyrosine phosphorylation and cysteine oxidation. Although these modifications share their reversibility depending on the intracellular environment, their effects on enzymatic activity are opposite, tyrosine phosphorylation being correlated to enzyme activation and thiol oxidation to complete inactivation. Several papers have suggested that both these modifications occur in response to the same stimuli i.e. cell proliferation induced by numerous growth factors and cytokines. Conversely, the possibility that these two regulation mechanisms act simultaneously on PTPs has not been established and very few reports investigated this dual regulation of PTPs. To underline the relevance of the question, we discuss several possibilities: (i) that tyrosine phosphorylation and cysteine oxidation of PTPs may share the same target molecules but with different kinetics; (ii) that PTP phosphorylation and oxidation may take place on different subcellular pools of the same protein and (iii) that these two modifications, although having divergent effects on enzyme activity, cooperate in the integrated and coordinated function of PTPs during receptor tyrosine kinase signalling. We believe that our perspective will open new perspectives on an ancient problem--the apparent contradiction of opposing enzymatic regulation of many PTPs--thus clarifying their role as positive or negative transducers (or both) of many extracellular stimuli.


Subject(s)
Cysteine/metabolism , Protein Processing, Post-Translational/physiology , Protein Tyrosine Phosphatases/metabolism , Tyrosine/metabolism , Enzyme Activation , Humans , Models, Biological , Oxidation-Reduction , Phosphorylation , Reactive Oxygen Species/metabolism
5.
Mol Biol Cell ; 16(1): 73-83, 2005 Jan.
Article in English | MEDLINE | ID: mdl-15525682

ABSTRACT

Cellular behavior can be considered to be the result of a very complex spatial and temporal integration of intracellular and extracellular signals. These signals arise from serum-soluble factors as well as from cell-substrate or cell-cell interactions. The current approach in mitogenesis studies is generally to analyze the effect of a single growth factor on serum-starved cells. In this context, a metabolic hormone such as insulin is found to be a mitogenic agent in many cellular types. In the present study, we have considered the effect of insulin stimulation in platelet-derived growth factor (PDGF)-activated NIH-3T3 and C2C12 cells. Our results show that insulin is able to inhibit strongly both NIH-3T3 and C2C12 cell growth induced by PDGF, one of the most powerful mitotic agents for these cell types. This inhibitory effect of insulin is due primarily to a premature down-regulation of the PDGF receptor. Thus, when NIH-3T3 or C2C12 cells are stimulated with both PDGF and insulin, we observe a decrease in PDGF receptor phosphorylation with respect to cells treated with PDGF alone. In particular, we find that costimulation with insulin leads to a reduced production of H2O2 with respect to cell stimulation with PDGF alone. The relative low concentration of H2O2 in PDGF/insulin-costimulated cell leads to a limited down-regulation of protein tyrosine phosphatases, and, consequently, to a reduced PDGF receptor phosphorylation efficiency. The latter is very likely to be responsible for the insulin-dependent inhibition of PDGF-receptor mitogenic signaling.


Subject(s)
Insulin/metabolism , Platelet-Derived Growth Factor/metabolism , Animals , Anti-Infective Agents, Local/pharmacology , Cell Communication , Cell Line , Cell Proliferation , Culture Media, Serum-Free/pharmacology , Down-Regulation , Endocytosis , Gentian Violet/pharmacology , Hydrogen Peroxide/pharmacology , Immunoprecipitation , Mice , Mitosis , NIH 3T3 Cells , Oxidation-Reduction , Phosphatidylinositol 3-Kinases/metabolism , Phosphorylation , Reactive Oxygen Species , Receptor, Insulin/metabolism , Receptors, Platelet-Derived Growth Factor/metabolism , Thymidine/pharmacology , Time Factors , Tyrosine/chemistry , Tyrosine/metabolism , src-Family Kinases/metabolism
7.
Cell Mol Life Sci ; 61(14): 1775-84, 2004 Jul.
Article in English | MEDLINE | ID: mdl-15241553

ABSTRACT

An acylphosphatase (AcPase) overexpression study was carried out on SH-SY5Y neuroblastoma cells, using a green fluorescent fusion protein (AcP-GFP), with GFP acting as a reporter protein. The cellular proliferation rate was significantly reduced by overexpression of AcPase by a factor of ten. In contrast, clones transfected with two inactive AcPase mutants showed a growth rate comparable to control cells. This suggests that AcPase catalyzes the proliferative down-regulation. AcPase-overexpressing clones showed a physiological mortality rate as assessed by an MTT reduction test and by evaluation of necrotic markers. DNA fragmentation analysis and assays of caspase-3 and poly (ADP-ribose) polymerase (PARP)-active fragments showed no evidence of any apoptotic pattern. AcPase overexpression led to a marked increase in PARP activity as well as Bcl-2 content; these are commonly up-regulated during differentiative processes in neuronal cells. In fact, the typical differentiation marker, growth-associated-protein 43, was significantly up-regulated. Microscopic observations also showed a clear increase in the differentiative phenotype in AcPase-overexpressing cells. Our results clearly show that AcPase plays a primary causative role in neuronal differentiation.


Subject(s)
Acid Anhydride Hydrolases/genetics , Cell Differentiation/physiology , Neuroblastoma/metabolism , Acid Anhydride Hydrolases/biosynthesis , Apoptosis/physiology , Biomarkers , Cell Division , Genes, Reporter , Oxidation-Reduction , Recombinant Fusion Proteins/genetics , Recombinant Fusion Proteins/metabolism , Acylphosphatase
8.
Cell Mol Life Sci ; 61(7-8): 982-91, 2004 Apr.
Article in English | MEDLINE | ID: mdl-15095018

ABSTRACT

Protein aggregation is a notable feature of various human disorders, including Parkinson's disease, Alzheimer's disease and many others systemic amyloidoses. An increasing number of observations in vitro suggest that transition metals are able to accelerate the aggregation process of several proteins found in pathological deposits, e.g. alpha-synuclein, amyloid beta (Abeta) peptide, beta(2)-microglobulin and fragments of the prion protein. Here we report the effects of metal ions on the aggregation rate of human muscle acylphosphatase, a suitable model system for aggregation studies in vitro. Among the different species tested, Cu(2+) produced the most remarkable acceleration of aggregation, the rate of the process being 2.5-fold higher in the presence of 0.1 mM metal concentration. Data reported in the literature suggest the possible role played by histidine residues or negatively charged clusters present in the amino acid sequence in Cu(2+)-mediated aggregation of pathological proteins. Acylphosphatase does not contain histidine residues and is a basic protein. A number of histidine-containing mutational variants of acylphosphatase were produced to evaluate the importance of histidine in the aggregation process. The Cu(2+)-induced acceleration of aggregation was not significantly altered in the protein variants. The different aggregation rates shown by each variant were entirely explained by the changes of hydrophobicity or propensity to form a beta structure introduced by the point mutation. The effect of Cu(2+) on acylphosphatase aggregation cannot therefore be attributed to the specific factors usually invoked in the aggregation of pathological proteins. The effect, rather, seems to be a general related to the chemistry of the polypeptide backbone and could represent an additional deleterious factor resulting from the alteration of the homeostasis of metal ions in cells.


Subject(s)
Acid Anhydride Hydrolases/chemistry , Acid Anhydride Hydrolases/metabolism , Copper/metabolism , Acid Anhydride Hydrolases/genetics , Acid Anhydride Hydrolases/ultrastructure , Animals , Humans , Models, Molecular , Mutation , Protein Conformation , Protein Denaturation , Acylphosphatase
9.
Cell Mol Life Sci ; 60(12): 2721-35, 2003 Dec.
Article in English | MEDLINE | ID: mdl-14685695

ABSTRACT

Cell differentiation is often associated with a block in the cell cycle. Growth factor signaling has been reported to be impaired in differentiated cells, due to the withdrawal of growth factors or to transcriptional down-regulation of their receptors. Our proposal is that the down regulation of growth factor signaling may be achieved through an alternative pathway: the decrease of growth factor receptor activation and the ensuing inhibition of intracellular pathways leading the cell to division. Here we report that platelet-derived growth factor receptor (PDGFr) signaling is down-regulated during muscle differentiation, although its expression level remains unchanged. PDGFr signaling inhibition is achieved through a decrease in the receptor tyrosine phosphorylation level, in particular of Tyr716, Tyr751, Tyr857 and Tyr1021, leading to down-regulation of intracellular signaling pathways. Furthermore, during myogenesis, the expression level of several phosphotyrosine phosphatases (PTPs) increases and most of them shift toward the reduced/activated state. We propose a causal link between the down-regulation of PDGFr tyrosine phosphorylation and the increases in PTP specific activity during myogenesis.


Subject(s)
Down-Regulation , Muscle Development/physiology , Receptors, Platelet-Derived Growth Factor/metabolism , Signal Transduction/physiology , Animals , Mice , Oxidation-Reduction , Phosphorylation , Protein Tyrosine Phosphatases/genetics , Protein Tyrosine Phosphatases/metabolism , Reactive Oxygen Species/metabolism
10.
Cell Mol Life Sci ; 59(6): 941-9, 2002 Jun.
Article in English | MEDLINE | ID: mdl-12169024

ABSTRACT

Low molecular weight protein tyrosine phosphatases (LMW-PTPs) are a family of 18-kDa enzymes involved in cell growth regulation. Despite very limited sequence similarity to the PTP superfamily, they display a conserved signature motif in the catalytic site. LMW-PTP associates and dephosphorylate many growth factor receptors, such as platelet-derived growth factor receptor (PDGF-r), insulin receptor and ephrin receptor, thus downregulating many of the tyrosine kinase receptor functions that lead to cell division. In particular, LMW-PTP acts on both growth-factor-induced mitosis, through dephosphorylation of activated PDGF-r, and on cytoskeleton rearrangement, through dephosphorylation of p190RhoGAP and the consequent regulation of the small GTPase Rho. LMW-PTP activity is modulated by tyrosine phosphorylation on two specific residues, each of them with specific characteristics. LMW-PTP activity on specific substrates depends also on its localization. Moreover, LMW-PTP is reversibly oxidized during growth factor signaling, leading to inhibition of its enzymatic activity. Recovery of phosphatase activity depends on the availability of reduced glutathione and involves the formation of an S-S bridge between the two catalytic site cysteines. Furthermore, studies on the redox state of LMW-PTP in contact-inhibited cells and in mature myoblasts suggest that LMW-PTP is a general and versatile modulator of growth inhibition.


Subject(s)
Isoenzymes/physiology , Protein Tyrosine Phosphatases/physiology , Animals , Cell Physiological Phenomena , Down-Regulation , Humans , Models, Molecular , Molecular Weight , Oxidation-Reduction , Phosphorylation , Structure-Activity Relationship , Up-Regulation
11.
Biochem Biophys Res Commun ; 296(3): 692-7, 2002 Aug 23.
Article in English | MEDLINE | ID: mdl-12176037

ABSTRACT

A profile-based search of the SWISS-PROT database reveals that most protein tyrosine phosphatases (PTPs) contain at least one caveolin-1-binding motif. To ascertain if the presence of caveolin-binding motif(s) in PTPs corresponds to their actual localization in caveolin-1-enriched membrane fractions, we performed subcellular fractionating experiments. We found that all tested PTPs (PTP1B, PTP1C, SHPTP2, PTEN, and LAR) are actually localized in caveolin-enriched membrane fractions, despite their distribution in other subcellular sites, too. More than 1/2 of LAR and about 1/4 of SHPTP2 and PTP-1C are localized in caveolin-enriched membrane fractions whereas, in these fractions, PTP-1B and PTEN are poorly concentrated. Co-immunoprecipitation experiments with antibodies specific for each tested PTP demonstrated that all five phosphatases form molecular complexes with caveolin-1 in vivo. Collectively, our findings propose that particular PTPs could perform some of their cellular actions or are regulated by recruitment into caveolin-enriched membrane fractions.


Subject(s)
Caveolins/metabolism , Membrane Microdomains/enzymology , Protein Tyrosine Phosphatases/metabolism , Amino Acid Motifs , Binding Sites , Caveolin 1 , Cell Line , Humans , Macromolecular Substances , Membrane Microdomains/metabolism , Precipitin Tests , Protein Transport , Protein Tyrosine Phosphatases/chemistry
12.
J Biol Chem ; 276(50): 46714-21, 2001 Dec 14.
Article in English | MEDLINE | ID: mdl-11590172

ABSTRACT

The folding of beta(2)-microglobulin (beta(2)-m), the protein forming amyloid deposits in dialysis-related amyloidosis, involves formation of a partially folded conformation named I(2), which slowly converts into the native fold, N. Here we show that the partially folded species I(2) can be separated from N by capillary electrophoresis. Data obtained with this technique and analysis of kinetic data obtained with intrinsic fluorescence indicate that the I(2) conformation is populated to approximately 14 +/- 8% at equilibrium under conditions of pH and temperature close to physiological. In the presence of fibrils extracted from patients, the I(2) conformer has a 5-fold higher propensity to aggregate than N, as indicated by the thioflavine T test and light scattering measurements. A mechanism of aggregation of beta(2)-m in vivo involving the association of the preformed fibrils with the fraction of I(2) existing at equilibrium is proposed from these results. The possibility of isolating and quantifying a partially folded conformer of beta(2)-m involved in the amyloidogenesis process provides new opportunities to monitor hemodialytic procedures aimed at the reduction of such species from the pool of circulating beta(2)-m but also to design new pharmaceutical approaches that consider such species as a putative molecular target.


Subject(s)
beta 2-Microglobulin/chemistry , beta 2-Microglobulin/metabolism , Benzothiazoles , Circular Dichroism , Coloring Agents/pharmacology , Congo Red/pharmacology , Electrophoresis, Capillary , Fluorescent Dyes/pharmacology , Humans , Hydrogen-Ion Concentration , Kinetics , Light , Microscopy, Electron , Models, Biological , Models, Chemical , Protein Conformation , Protein Denaturation , Protein Folding , Scattering, Radiation , Temperature , Thiazoles/pharmacology , Time Factors , Ultraviolet Rays
13.
J Biol Chem ; 276(52): 49156-63, 2001 Dec 28.
Article in English | MEDLINE | ID: mdl-11595742

ABSTRACT

Low molecular weight protein-tyrosine phosphatase (LMW-PTP) is an enzyme involved in mitogenic signaling and cytoskeletal rearrangement after platelet-derived growth factor (PDGF) stimulation. Recently, we demonstrated that LMW-PTP is regulated by a redox mechanism involving the two cysteine residues of the catalytic site, which turn reversibly from reduced to oxidized state after PDGF stimulation. Since recent findings showed a decrease of intracellular reactive oxygen species in contact inhibited cells and a lower tyrosine phosphorylation level in dense cultures in comparison to sparse ones, we studied if the level of endogenous LMW-PTP is regulated by growth inhibition conditions, such as cell confluence and differentiation. Results show that both cell confluence and cell differentiation up-regulate LMW-PTP expression in C2C12 and PC12 cells. We demonstrate that during myogenesis LMW-PTP is regulated at translational level and that the protein accumulates at the plasma membrane. Furthermore, we showed that both myogenesis and cell-cell contact lead to a dramatic decrease of tyrosine phosphorylation level of PDGF receptor. In addition, we observed an increased association of the receptor with LMW-PTP during myogenesis. Herein, we demonstrate that myogenesis decreases the intracellular level of reactive oxygen species, as observed in dense cultures. As a consequence, LMW-PTP turns from oxidized to reduced form during muscle differentiation, increasing its activity in growth inhibition conditions such as differentiation. These data suggest that LMW-PTP plays a crucial role in physiological processes, which require cell growth arrest such as confluence and differentiation.


Subject(s)
Cell Differentiation , Cell Division , Protein Tyrosine Phosphatases/metabolism , Animals , Becaplermin , Cell Count , Cell Line , Gene Expression Regulation, Developmental , Humans , Microscopy, Confocal , Molecular Weight , Muscle Development/physiology , Oxidation-Reduction , PC12 Cells , Phosphorylation , Platelet-Derived Growth Factor/metabolism , Protein Tyrosine Phosphatases/chemistry , Proto-Oncogene Proteins c-sis , Rats , Reactive Oxygen Species/metabolism , Receptors, Platelet-Derived Growth Factor/metabolism , Up-Regulation
14.
J Biol Chem ; 276(45): 41862-9, 2001 Nov 09.
Article in English | MEDLINE | ID: mdl-11551950

ABSTRACT

Acylphosphatase is expressed in vertebrates as two molecular forms, the organ common and the muscle types. The former does not contain cysteine residues, whereas the latter contains a single conserved cysteine (Cys-21). We demonstrated that H(2)O(2) at micromolar levels induces, in vitro, the formation of a disulfide dimer of muscle acylphosphatase, which displays properties differing from those of the reduced enzyme. In particular, we observed changes in the kinetic behavior of its intrinsic ATPase activity, whereas the kinetic behavior of its benzoyl phosphatase activity does not change. Moreover, the disulfide dimer is capable of interacting with some polynucleotides such as poly(G), poly(C), and poly(T) but not with poly(A), whereas the reduced enzyme does not bind polynucleotides. Experiments performed with H(2)O(2) in the presence of increasing SDS concentrations demonstrated that disulfide dimer formation is prevented by SDS concentrations higher than 300 microm, suggesting that a non-covalently-linked dimer is present in non-denaturing solvents. Light-induced cross-linking experiments performed on the Cys-21 --> Ser mutant in the pH range 3.8-9.0 have demonstrated that a non-covalently-linked dimer is in fact present in non-denaturing solutions and that an enzyme group with a pK(a) of 6.4 influences the monomer-dimer equilibrium.


Subject(s)
Acid Anhydride Hydrolases/chemistry , Disulfides/chemistry , Hydrogen Peroxide/pharmacology , Muscles/enzymology , Acid Anhydride Hydrolases/physiology , Adenosine Triphosphatases/metabolism , Dimerization , Hydrogen-Ion Concentration , Poly A/metabolism , Acylphosphatase
15.
Acta Neurochir (Wien) ; 143(5): 457-63; discussion 463-4, 2001.
Article in English | MEDLINE | ID: mdl-11482695

ABSTRACT

BACKGROUND: Carotid endarterectomy has been reported to increase the time free from cerebral ischemic events in both symptomatic and asymptomatic patients with a high grade of stenosis of the internal carotid artery. In cases in whom the compensatory circulation during the carotid clamp time is not sufficient, the use of intraluminal shunts has been proposed. However, the use of intraluminal shunts present several problems, such as the technical difficulties in positioning the shunt, the variability of time requested for the placement, the inconstancy of the blood flow during surgery, and the need to clamp off the carotid to introduce and remove the shunt. For these reasons, most operators prefer not to employ intraluminal shunts, while others do use them only in selected cases. The purpose of this work is to present, for the first time, a new type of temporary extraluminal shunt, connecting the femoral to the internal carotid artery with the interposition of a roller pump to regulate the blood flow. This method allows one to perform carotid endarterectomy without interrupting the blood flow to the brain. METHODS: 407 consecutive patients, who underwent carotid endarterectomy between August 1992 and April 2000, were considered. 35 patients presented an absolutely insufficient collateral circulation, demonstrated by important modifications of the electroencephalographic monitoring during the carotid clamp time. In these patients the endarterectomy was performed using a new femoral-carotid extraluminal shunt. FINDINGS: In all the cases in whom the femoral-carotid extraluminal shunt was positioned, the normalisation of electroencephalographic monitoring was achieved by regulating the blood flow with the interposed roller pump. The use and the placement of the shunt was simple and easy. None of the patients showed postoperative complications, except for one who had a stroke two days after surgery. INTERPRETATION: The results obtained, although to be confirmed by further studies, seem to demonstrate the effectiveness of our femoral-carotid extraluminal shunt, which was simple to use and safe.


Subject(s)
Brain Ischemia/prevention & control , Carotid Artery, Internal/surgery , Carotid Stenosis/surgery , Endarterectomy, Carotid/methods , Femoral Artery/surgery , Aged , Aged, 80 and over , Anastomosis, Surgical , Brain/physiopathology , Brain Ischemia/physiopathology , Carotid Stenosis/physiopathology , Cerebrovascular Circulation , Collateral Circulation , Electroencephalography , Endarterectomy, Carotid/adverse effects , Female , Humans , Male , Middle Aged , Treatment Outcome
16.
J Biol Chem ; 276(40): 37149-54, 2001 Oct 05.
Article in English | MEDLINE | ID: mdl-11479314

ABSTRACT

The native state of human muscle acylphosphatase (AcP) presents two alpha-helices. In this study we have investigated folding and aggregation of a number of protein variants having mutations aimed at changing the propensity of these helical regions. Equilibrium and kinetic measurements of folding indicate that only helix-2, spanning residues 55-67, is largely stabilized in the transition state for folding therefore playing a relevant role in this process. On the contrary, the aggregation rate appears to vary only for the variants in which the propensity of the region corresponding to helix-1, spanning residues 22-32, is changed. Mutations that stabilize the first helix slow down the aggregation process while those that destabilize it increase the aggregation rate. AcP variants with the first helix destabilized aggregate with rates increased to different extents depending on whether the introduced mutations also alter the propensity to form beta-sheet structure. The fact that the first alpha-helix is important for aggregation and the second helix is important for folding indicates that these processes are highly specific. This partitioning does not reflect the difference in intrinsic alpha-helical propensities of the two helices, because helix-1 is the one presenting the highest propensity. Both processes of folding and aggregation do not therefore initiate from regions that have simply secondary structure propensities favorable for such processes. The identification of the regions involved in aggregation and the understanding of the factors that promote such a process are of fundamental importance to elucidate the principles by which proteins have evolved and for successful protein design.


Subject(s)
Acid Anhydride Hydrolases/chemistry , Muscles/enzymology , Acid Anhydride Hydrolases/genetics , Acid Anhydride Hydrolases/metabolism , Enzyme Stability , Humans , Kinetics , Models, Molecular , Mutation , Protein Folding , Protein Structure, Secondary , Acylphosphatase
17.
J Biol Chem ; 276(36): 33478-87, 2001 Sep 07.
Article in English | MEDLINE | ID: mdl-11429404

ABSTRACT

Low molecular weight protein tyrosine phosphatase (LMW-PTP) is an enzyme involved in platelet-derived growth factor (PDGF)-induced mitogenesis and cytoskeleton rearrangement because it is able to bind and dephosphorylate the activated receptor. LMW-PTP presents two cysteines in positions 12 and 17, both belonging to the catalytic pocket; this is a unique feature of LMW-PTP among all protein tyrosine phosphatases. Our previous results demonstrated that in vitro LMW-PTP is oxidized by either H(2)O(2) or nitric oxide with the formation of a disulfide bond between Cys-12 and Cys-17. This oxidation leads to reversible enzyme inactivation because treatment with reductants permits catalytic activity rescue. In the present study we investigated the in vivo inactivation of LMW-PTP by either extracellularly or intracellularly generated H(2)O(2), evaluating its action directly on its natural substrate, PDGF receptor. LMW-PTP is oxidized and inactivated by exogenous oxidative stress and recovers its activity after oxidant removal. LMW-PTP is oxidized also during PDGF signaling, very likely upon PDGF-induced H(2)O(2) production, and recovers its activity within 40 min. Our results strongly suggest that reversibility of in vivo LMW-PTP oxidation is glutathione-dependent. In addition, we propose an intriguing and peculiar role of Cys-17 in the formation of a S-S intramolecular bond, which protects the catalytic Cys-12 from further and irreversible oxidation. On the basis of our results we propose that the presence of an additional cysteine near the catalytic cysteine could confer to LMW-PTP the ability to rapidly recover its activity and finely regulate PDGF receptor activation during both extracellularly and intracellularly generated oxidative stress.


Subject(s)
Cysteine/chemistry , Isoenzymes , Oxidation-Reduction , Protein Tyrosine Phosphatases/metabolism , Proto-Oncogene Proteins , Receptors, Platelet-Derived Growth Factor/metabolism , 3T3 Cells , Animals , Blotting, Northern , Blotting, Western , Catalysis , Cell Line , Culture Media, Serum-Free/metabolism , Enzyme Activation , Glutathione/chemistry , Humans , Hydrogen Peroxide/pharmacology , Mice , Mutagenesis, Site-Directed , Mutation , Oxidative Stress , Oxygen/metabolism , Phosphorylation , Precipitin Tests , Protein Binding , Reactive Oxygen Species/metabolism , Time Factors , Transfection , Tyrosine/metabolism
18.
Electrophoresis ; 22(3): 576-85, 2001 Feb.
Article in English | MEDLINE | ID: mdl-11258771

ABSTRACT

Small tyrosine phoshatase 1 (Stp1) is a Schizosaccharomyces pombe low-molecular-mass phosphotyrosine-phosphatase 50% identical to Saccharomyces cerevisiae Ltp1. In order to investigate the role of Stp1 in yeast, a mutant was generated having the characteristic of a dominant negative molecule. Changes in protein tyrosine phosphorylation in S. cerevisiae proteome in response to Stp1 or its dominant negative mutant expression were analyzed by high-resolution two-dimensional (2-D) electrophoresis. The most remarkable result is the modification by phosphorylation on tyrosine of several proteins involved in carbohydrate metabolism. Twelve proteins were identified on the basis of their positions in the anti-phosphotyrosine immunoblot of the 2-D electrophoresis. Ten of these present tyrosyl residues that are within the consensus sequence for protein kinase CK2 (casein kinase-2). These data open the possibility for the identification of Stp1 substrates in yeast and provide hints about the nature of tyrosine phosphorylating agents in yeast and in other organisms where bona fide tyrosine kinases are lacking.


Subject(s)
Protein Tyrosine Phosphatases/metabolism , Saccharomyces cerevisiae/enzymology , Acrylic Resins , Amino Acid Sequence , Cloning, Molecular , Culture Media , Electrophoresis, Gel, Two-Dimensional/methods , Gene Expression , Genes, Fungal , Molecular Sequence Data , Phosphorylation , Plasmids , Protein Tyrosine Phosphatases/genetics , Saccharomyces cerevisiae/growth & development , Silver Staining , Tyrosine/metabolism
19.
Protein Sci ; 10(4): 879-86, 2001 Apr.
Article in English | MEDLINE | ID: mdl-11274479

ABSTRACT

It is known that human muscle acylphosphatase (AcP) is able, under appropriate conditions in vitro, to aggregate and form amyloid fibrils of the type associated with human diseases. A number of compounds were tested for their ability to bind specifically to the native conformation of AcP under conditions favoring denaturation and subsequent aggregation and fibril formation. Compounds displaying different binding affinities for AcP were selected and their ability to inhibit protein fibrillization in vitro was evaluated. We found that compounds displaying a relatively high affinity for AcP are able to significantly delay protein fibrillization, mimicking the effect of stabilizing mutations; in addition, the effectiveness of such outcome correlates positively to both ligand concentration and affinity to the native state of AcP. By contrast, the inhibitory effect of ligands on AcP aggregation disappears in a mutant protein in which such binding affinity is lost. These results indicate that the stabilization of the native conformation of amyloidogenic proteins by specific ligand binding can be a strategy of general interest to inhibit amyloid formation in vivo.


Subject(s)
Acid Anhydride Hydrolases/chemistry , Acid Anhydride Hydrolases/metabolism , Amyloid/chemistry , Amyloid/metabolism , Circular Dichroism , Escherichia coli/enzymology , Escherichia coli/genetics , In Vitro Techniques , Kinetics , Mutation/genetics , Protein Conformation , Protein Denaturation , Acylphosphatase
20.
J Biol Chem ; 276(22): 18849-54, 2001 Jun 01.
Article in English | MEDLINE | ID: mdl-11279120

ABSTRACT

Low M(r) phosphotyrosine-protein phosphatase is involved in the regulation of several tyrosine kinase growth factor receptors. The best characterized action of this enzyme is on the signaling pathways activated by platelet-derived growth factor, where it plays multiple roles. In this study we identify tyrosine-phosphorylated caveolin as a new potential substrate for low M(r) phosphotyrosine-protein phosphatase. Caveolin is tyrosine-phosphorylated in vivo by Src kinases, recruits into caveolae, and hence regulates the activities of several proteins involved in cellular signaling cascades. Our results demonstrate that caveolin and low M(r) phosphotyrosine-protein phosphatase coimmunoprecipitate from cell lysates, and that a fraction of the enzyme localizes in caveolae. Furthermore, in a cell line sensitive to insulin, the overexpression of the C12S dominant negative mutant of low M(r) phosphotyrosine-protein phosphatase (a form lacking activity but able to bind substrates) causes the enhancement of tyrosine-phosphorylated caveolin. Insulin stimulation of these cells induces a strong increase of caveolin phosphorylation. The localization of low M(r) phosphotyrosine-protein phosphatase in caveolae, the in vivo interaction between this enzyme and caveolin, and the capacity of this enzyme to rapidly dephosphorylate phosphocaveolin, all indicate that tyrosine-phosphorylated caveolin is a relevant substrate for this phosphatase.


Subject(s)
Caveolins/metabolism , Protein Tyrosine Phosphatases/metabolism , Tyrosine/metabolism , 3T3 Cells , Animals , Caveolin 1 , Cell Line , Cell Membrane/metabolism , DNA, Complementary/metabolism , Genes, Dominant , Humans , Mice , Phosphorylation , Precipitin Tests , Recombinant Proteins/metabolism , Signal Transduction , Time Factors , Transfection
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