Vascular disease and heart failure impart an enormous burden in terms of global morbidity and mortality. Although there are many different causes of cardiac and vascular disease, most share an important pathological mechanism: oxidative stress. In the failing heart, oxidative stress occurs in the myocardium and correlates with left ventricular dysfunction. Reactive oxygen species (ROS) negatively affect myocardial calcium handling, cause arrhythmias, and contribute to cardiac remodeling by inducing hypertrophic signaling, apoptosis, and necrosis. Similarly, oxidative balance in the vasculature is tightly regulated by a wealth of pro- and anti-oxidant systems that orchestrate region-specific ROS production and removal. ROS also regulate multiple vascular cell functions, including: endothelial and smooth muscle cell growth, proliferation, and migration; angiogenesis; apoptosis; vascular tone; host defenses; and genomic stability. However, excessive levels of ROS promote vascular disease via direct and irreversible oxidative damage to macromolecules, as well as disruption of redox-dependent vascular wall signaling processes.

Low molecular weight phosphotyrosine-protein phosphatase (LMW-PTP) shares no general sequence homology with other PTPs, although it has an active site sequence motif CXXXXXR and a reaction mechanism identical to those of all PTPs. The main function of this enzyme is the down-regulation of platelet-derived growth factor and insulin receptors. Both human LMW-PTP isoenzymes are inactivated by H 2 O 2 . The enzymes are protected from inactivation by P i , a competitive inhibitor, suggesting that the H 2 O 2 reaction is directed to active site. Analysis of free thiols performed on the inactivated enzymes demonstrates that only two out of the eight LMW-PTP cysteines are modified. Time-course high performance liquid chromatography-electrospray mass spectrometry, together with specific radiolabeling and tryptic fingerprint analyses, enables us to demonstrate that H 2 O 2 causes the oxidation of Cys-12 and Cys-17 to form a disulfide bond. Because both residues are localized into the active site region, this modification inactivates the enzyme. Fluorescence spectroscopy experiments suggest that the fold of the enzyme is modified during oxidation by H 2 O 2 . Because a physiological concentration of H 2 O 2 produces enzyme inactivation and considering that the activity is restored by reduction with low molecular weight thiols, we suggest that oxidative stress conditions and other processes producing hydrogen peroxide regulate the LMW-PTP in the cell.Protein tyrosine phosphorylation in eucaryotes is a key mechanism for cellular control, because it is involved in several processes, such as cellular metabolism, proliferation, differentiation, and oncogenic transformation (1). A fine balancing of cellular protein tyrosine phosphorylation levels is determined by regulating the activities of protein-tyrosine kinases and/or protein-tyrosine phosphatases (PTPs).1 Receptor protein-tyrosine kinases are considered to be the major enzymes regulating mitogenic protein phosphorylation cascades; nevertheless, the presence of SH2 domains in particular PTPs and the receptorlike structure of some membrane PTPs clearly indicate that PTPs are also regulated in the cell. The PTP superfamily consists of four main families: the tyrosine-specific phosphatases, the VH1-like dual specificity phosphatases, the cdc25 phosphatases, and the low molecular weight phosphatases (LMW-PTPs). Despite extremely limited sequence similarity, all share an active site motif consisting of a cysteine and an arginine separated by five residues (CXXXXXR, where X is any amino acid). All PTPs have identical catalytic mechanism, which involves the formation of a cysteinyl-phosphate intermediate (2).Recent papers from our laboratory have demonstrated that LMW-PTP is involved in the regulation of cellular signaling started by the activation of PDGF and insulin receptors (3-5). In fact, the overexpression of the wild type enzyme in NIH/3T3 cells causes decrease of cellular growth rate and of phosphorylation level of the PDGF receptor (3). Furthermore, the overexpression in ...

A new phytotoxic protein (cerato-platanin) of about 12.4 kDa has been identified in culture filtrates of the Ascomycete Ceratocystis fimbriata f. sp. platani, the causal agent of canker stain disease. The toxicity of the pure protein was bioassayed by detecting the inducing necrosis in tobacco leaves. The pure protein also elicited host synthesis of fluorescent substances in tobacco and plane (Platanus acerifolia) leaves. We purified the protein from culture medium to homogeneity. Its complete amino acid sequence was determined; this protein consists of 120 amino acid residues, contains 4 cysteines (S-S-bridged), and has a high percentage of hydrophobic residues. The molecular weight calculated from the amino acid sequence agrees with that determined by mass spectrometry, suggesting that no post-transnational modification occurs. Searches performed by the BLAST program in data banks (Swiss-Prot, EBI, and GenBank) revealed that this protein is highly homologous with two proteins produced by other Ascomycete fungi. One, produced during infection of wheat leaves, is codified by the snodprot1 gene of Phaeosphaeria nodorum (the causal agent of glume blotch of wheat), whereas the other is the rAsp f13 allergen from Aspergillus fumigatus. Furthermore, the N terminus of cerato-platanin is homologous with that of cerato-ulmin, a phytotoxic protein belonging to the hydrophobin family and produced by Ophiostoma (Ceratocystis) ulmi, a fungus responsible for Dutch elm disease.The European plane tree (Platanus acerifolia) is an ornamental plant species of the urban environment. A great number of plane trees in the parks and towns of southern Europe have been destroyed by Ceratocystis fimbriata (Ell. and Halst.) Davidson f. sp. platani Walter, the Ascomycete responsible for canker stain disease (1). This disease is characterized by foliar wilting and spreading lesions that involve phloem, cambium, and extensive regions of sapwood (2, 3). The pathogen spreads from tree to tree by means of root grafts of closely spaced plants and, more frequently, through wounds caused by pruning (4).The American species Platanus occidentalis has been shown to contain a source of resistance to C. fimbriata f. sp. platani that could prove of great interest in the genetic improvement of the European plane (5). Known post-infection host defense mechanisms involve physical factors such as the occlusion of the xylematic vessels and the compartmentalization of infected tissue areas as well as the production of flavans, umbelliferone, and scopoletine phytoalexins (6 -9). Unfortunately, only resistant P. occidentalis clones, not yet acclimatized to Europe, localized the disease (7,8). Recent papers (10, 11) have shown that C. fimbriata f. sp. platani displays an array of phytotoxic metabolites possibly involved in determining some of the symptoms of canker stain.In the present paper we report on the purification procedure, the amino acid sequence, and the characterization of the biological activity of a new protein (named cerato-platanin) from the cul...

Increased production of reactive oxygen species (ROS) and loss of endothelial NO bioavailability are key features of vascular disease in diabetes mellitus. The p66 Shc adaptor protein controls cellular responses to oxidative stress. Mice lacking p66 Shc (p66 Shc؊/؊ ) have increased resistance to ROS and prolonged life span. The present work was designed to investigate hyperglycemia-associated changes in endothelial function in a model of insulin-dependent diabetes mellitus p66 Shc؊/؊ mouse. p66 Shc؊/؊ and wild-type (WT) mice were injected with citrate buffer (control) or made diabetic by an i.p. injection of 200 mg of streptozotocin per kg of body weight. Streptozotocin-treated p66 Shc؊/؊ and WT mice showed a similar increase in blood glucose. However, significant differences arose with respect to endothelial dysfunction and oxidative stress. WT diabetic mice displayed marked impairment of endotheliumdependent relaxations, increased peroxynitrite (ONOO ؊ ) generation, nitrotyrosine expression, and lipid peroxidation as measured in the aortic tissue. In contrast, p66 Shc؊/؊ diabetic mice did not develop these high-glucose-mediated abnormalities. Furthermore, protein expression of the antioxidant enzyme heme oxygenase 1 and endothelial NO synthase were up-regulated in p66 Shc؊/؊ but not in WT mice. We report that p66 Shc؊/؊ mice are resistant to hyperglycemia-induced, ROS-dependent endothelial dysfunction. These data suggest that p66 Shc adaptor protein is part of a signal transduction pathway relevant to hyperglycemia vascular damage and, hence, may represent a novel therapeutic target against diabetic vascular complications.