The increased iron deposition in legs affected by CVD seems to be more instable in ulcer patients, leading to iron release in the serum of the affected leg. Our data suggest the iron-driven pathway as a further mechanism for MMP hyperexpression leading to tissue lesion.

Determination of presence of hemosiderin in the urine is a new, sensitive, cost-effective, noninvasive, and repeatable test that enables detection of substantial microcirculatory overload in patients with CVI.

Purified bile salt hydrolase from bile-adapted Xanthomonas maltophilia displays Michaelis-Menten kinetics on cholylglycine and cholyltaurine and hydrolyzes bile salts also in crude bovine bile. The protein is a dimer and is resistant to proteinases and to heating at 55 to 60°C for up to 60 min, in agreement with calorimetric data.Recently, we examined the metabolism of bile salts by the bile-adapted strain Xanthomonas maltophilia CBS 827.97, which hydrolyzes bile salt conjugates, rearranging the steroid nucleus (1, 5). Bile salts have multiple functions in digestion of lipids, solubilization, and excretion of cholesterol (2,19,23). Ursodeoxycholic acid is particularly interesting in this regard, solubilizing gallbladder cholesterol stones after oral administration (20). Ursodeoxycholic acid is currently prepared by alkaline hydrolysis of bile conjugates, inversion of the hydroxyl configuration at carbon 7, and resolution of the racemic mixture (reviewed in reference 1). Procedures for enzymatic synthesis would be useful for controlling stereochemistry, increasing yields, and avoiding toxic or aggressive chemicals. In this paper we describe a solution to the hydrolysis step, in which purified bile salt hydrolase (cholylglycine hydrolase [CGH], EC 3.5.1.24) from our bacterial strain is used.Procedures for purification of CGH and of other enzymes of bile salt metabolism are covered by an Italian patent (1). For purification of CGH, the enzyme was isolated from 10 g of bacteria grown as described previously (5) and lysed by overnight stirring in 10 volumes of 20 mM sodium phosphate-1 mM EDTA-2 mM mercaptoethanol (pH 7.5) (NaP buffer) containing 0.3 mg of egg lysozyme per ml and 1 mM phenylmethylsulfonyl fluoride. Cell debris was removed by centrifugation (200,000 ϫ g, 20 min), and nucleic acids were removed by precipitation with protamine sulfate (2 mg/ml). The supernatant, adjusted to pH 8.3, was chromatographed on a DEAESepharose column (30 ml) and eluted with a linear 0 to 0.25 M NaCl gradient in NaP buffer (pH 8.3). The second protein peak, displaying hydrolase activity, was fractionated with ammonium sulfate in order to collect proteins insoluble between 45 and 75% saturation. After dialysis against NaP buffer, the phosphate concentration of the solution was adjusted to 200 mM at pH 7.5 by adding solid salts, and the solution was heated to 55°C with continuous swirling in a water bath at 70°C. After the solution was cooled on ice, denatured aggregated proteins were removed by high-speed centrifugation, and the supernatant, diluted with 1 volume of water, was loaded onto a phenyl-Sepharose column (2 ml) equilibrated in 100 mM phosphate buffer (pH 7.5). Hydrolase activity was eluted with NaP buffer. The CGH activity assay was performed by measuring glycine release from 5 mM cholylglycine in NaP buffer by a cadmium-ninhydrin procedure (6). For the cholyltaurine hydrolysis assay, we employed a different ninhydrin formulation (17). The protein concentration was determined as described previously (18).The yield was 0...

The aim of the present study was to analyze the protein composition of ductal breast carcinoma and the surrounding normal tissue in individual patients using comparative 2D proteomics and mass spectrometry to detect candidate disease biomarkers for diagnosis and prognosis. Samples of normal and cancerous tissue obtained form 28 patients were analyzed. Chaperonins and cytoskeletal proteins predominated among the 11 proteins for which major changes in abundance were detected. Of these 11 proteins with an altered expression, 2 had a decreased expression and 9 had an increased expression. In addition, the abundance of a few cytokeratins was also altered; however, they were not capable of serving as specific circulatory biomarkers. The proteins which we observed to exhibit an altered expression in infiltrating ductal breast carcinoma may be exploited as novel targets for therapeutic interventions or represent novel diagnostic/prognostic markers for the early detection of aggressive tumors, particularly those with multridrug-resistant phenotypes during the earlier stages of the disease.

Activation of tissue transglutaminase by calcium involves a conformational change which allows exposition of the active site to the substrate via movements of domains 3 and 4 that lead to an increase of the inter-domain distance. The inhibitor GTP counteracts these changes. Here we investigate the possible existence of non-native conformational states still compatible with the enzyme activity produced by chemical and thermal perturbations. The results indicate that chemical denaturation is reversible at low guanidine concentrations but irreversible at high concentrations of guanidine. Indeed, at low guanidine concentrations tissue TG-ase exists in a non-native state which is still affected by the ligands as in the native form. In contrast, thermal unfolding is always irreversible, with aggregation and protein self-crosslinkage in the presence of calcium. DSC thermograms of the native protein in the absence of ligands consist of two partly overlapped transitions, which weaken in the presence of calcium and merge together and strengthen in the presence of GTP. Overall, the present work shows, for the first time, the reversible denaturation of a TG-ase isoenzyme and suggests the possibility that also in in vivo, the enzyme may acquire non-native conformations relevant to its patho-physiological functions.