Isolation, characterization, and nuclease activity of biologically relevant chromium(V) complexes with monosaccharides and model diols. Likely intermediates in chromium-induced cancers.

The stabilization of Cr(V) by biological 1,2-diolato ligands, including carbohydrates, glycoproteins, and sialic acid derivatives, is likely to play a crucial role in the genotoxicity of Cr(VI) and has also been implicated in the antidiabetic effect of Cr(III). Previously, such complexes have been observed by electron paramagnetic resonance (EPR) spectroscopy in living cells or animals, treated with carcinogenic Cr(VI), as well as in numerous model systems, but attempts to isolate them have been elusive. Recently, the first crystal structure of a Cr(V) complex with cis-1,2-cyclohexanediol (1, a close structural analogue of carbohydrates) has been reported. In this work, Cr(V) complexes of the general formula [Cr(V)OL2](-) [where LH2 = 1, cis-1,2-cyclopentanediol (2), D-glucose (3), D-mannose (4), D-galactose (5), and D-ribose (6)] have been isolated from light-catalyzed reactions of Cr(VI) (anhydrous Na2Cr2O7) with slight molar excesses of the corresponding ligands in N,N-dimethylformamide. The complexes were characterized by elemental analyses, electrospray mass spectrometry (ESMS), and EPR spectroscopy. Studies by electronic absorption spectroscopy have shown that the solids isolated from reactions of Cr(VI) with 3-6 contained mixtures of Cr(V) complexes (40-65 mol %) and Cr(III) species (probably complexes with oxidized ligands), while those from reactions with 1 and 2 were practically pure Cr(V). The first isolation of solids containing significant proportions of chromium(V) monosaccharide complexes led to the definitive assignment of their general formula ([Cr(V)OL2](-), based on ESMS), in agreement with the earlier EPR spectroscopic data. The first direct comparison of the decomposition rates of Cr(V) complexes with 1-6, made possible by isolation of the solids, have shown that the complexes with five-membered-ring ligands (2 and 6) are more stable at pH ∼ 7 compared with their six-membered-ring counterparts (1 and 3-5). This finding emphasizes the likely biological roles of chromium(V) pentose complexes, e.g., those with sugar residues of RNA, ATP, or NAD(P)H. Finally, the first direct evidence for the ability of these Cr(V) complexes to cause oxidative DNA damage in the absence of added reductants or oxidants has been obtained. These data support significant roles for chromium(V) 1,2-diolato complexes in the diverse biological activities of Cr(VI) and Cr(III).

Identification of novel diagnostic biomarkers for asthma and chronic obstructive pulmonary disease.

RATIONALE: Proteomics may identify a useful panel of biomarkers for identification of asthma and chronic obstructive pulmonary disease (COPD). OBJECTIVES: To conduct an unsupervised analysis of peripheral blood proteins in well-characterized subjects with asthma and COPD, and identify and validate a biomarker panel for disease discrimination. METHODS: Two-dimensional difference gel electrophoresis was used to separate plasma proteins from healthy control subjects, stable patients with asthma, and individuals with COPD. Candidate protein markers were identified by matrix assisted laser desorption ionization time of flight mass spectrometry and subsequently validated in two populations via immunoassay. A panel of four biomarkers was selected and their ability to distinguish between groups was assessed in isolation and in combination in two separate validation populations. MEASUREMENTS AND MAIN RESULTS: Seventy-two protein spots displayed significantly different expression levels between the three subject groupings (P < 0.05). Fifty-eight were positively identified, representing 20 unique proteins. A panel of four biomarkers (α(2)-macroglobulin, haptoglobin, ceruloplasmin, and hemopexin) was able to discriminate with statistical significance between the clinical groups of patients with asthma, patients with COPD, and control subjects, and these results were confirmed in a second clinical population of older adults with airflow obstruction. CONCLUSIONS: Proteomics has identified novel biomarkers for asthma and COPD, and shown that the iron metabolism pathways and acute-phase response may be involved in the pathogenesis of airway disease. The panel of peripheral blood biomarkers has the potential to become an extremely useful addition to the clinical diagnosis and management of respiratory disease.

Sialoglycoprotein and carbohydrate complexes in chromium toxicity.

Chromium(VI) compounds are amongst the most widely encountered industrial carcinogens and are of increasing concern with respect to environmental exposure. Sialoglycoproteins and carbohydrates play a crucial role in stabilizing oxoCr(V) intermediates, which are produced by extracellular and intracellular reduction of chromium(VI). Recent research has addressed the molecular characterization of oxoCr(V)-sialoglycoprotein and -carbohydrate complexes and the roles that these species may play in Cr(VI) metabolism and carcinogenesis. Particular highlights include the role of oxoCr(V) complexes of extracellular sialoglycoproteins, intracellular D-glucose, and related species and their potential roles in Cr(VI)-induced genotoxicity.

Isolation, identification and characterisation of starch-interacting proteins by 2-D affinity electrophoresis.

A 2-D affinity electrophoretic technique (2-DAE) has been used to isolate proteins that interact with various starch components from total barley endosperm extracts. In the first dimension, proteins are separated by native PAGE. The second-dimensional gel contains polysaccharides such as amylopectin and glycogen. The migration of starch-interacting proteins in this dimension is determined by their affinity towards a particular polysaccharide and these proteins are therefore spatially separated from the bulk of proteins in the crude extract. Four distinct proteins demonstrate significant affinity for amylopectin and have been identified as starch branching enzyme I (SBEI), starch branching enzyme IIa (SBEIIa), SBEIIb and starch phosphorylase using polyclonal antibodies and zymogram activity analysis. In the case of starch phosphorylase, a protein spot was excised from a 2-DAE polyacrylamide gel and analysed using Q-TOF MS/MS, resulting in the alignment of three internal peptide sequences with the known sequence of the wheat plastidic starch phosphorylase isoform. This assignment was confirmed by the determination of the enzyme's function using zymogram analysis. Dissociation constants (Kd) were calculated for the three enzymes at 4 degrees C and values of 0.20, 0.21 and 1.3 g/L were determined for SBEI, SBEIIa and starch phosphorylase, respectively. Starch synthase I could also be resolved from the other proteins in the presence of glycogen and its identity was confirmed using a polyclonal antibody and by activity analysis. The 2-DAE method described here is simple, though powerful, enabling protein separation from crude extracts on the basis of function.