Mechanistic Insight into the Catechol Oxidase Activity by a Biomimetic Dinuclear Copper Complex

The biomimetic catalytic oxidation of 3,5-ditert- butylcatechol by the dicopper(II) complex of the ligand a,a¢-bis{bis[1-(1¢-methyl-2¢-benzimidazolyl) methyl]amino}-m-xylene in the presence of dioxygen has been investigated as a function of temperature and pH in a mixed aqueous/organic solvent. The catalytic cycle occurs in two steps, the first step being faster than the second step. In the first step, one molecule of catechol is oxidized by the dicopper(II) complex, and the copper(II) centers are reduced. From the pH dependence, it is deduced that the active species of the process is the monohydroxo form of the dinuclear complex. In the second step, the second molecule of catechol is oxidized by the dicopper(I)-dioxygen complex formed upon oxygenation of the reduced complex. In both cases, catechol oxidation is an inner-sphere electron transfer process involving binding of the catechol to the active species. The binary catechol-dicopper(II) complex formed in the first step could be characterized at very low temperature (90 C), where substrate oxidation is blocked. On the contrary, the ternary complex of dicopper( I)-O2-catechol relevant to the second step does not accumulate in solution and could not be characterized, even at low temperature. The investigation of the biphasic kinetics of the catalytic reaction over a range of temperatures allowed the thermodynamic (DH and DS) and activation parameters (DH „ and DS „ ) connected with the key steps of the catecholase process to be obtained

The metal-nonoate Ni(SalPipNONO) inhibits in vitro tumor growth, invasiveness and angiogenesis

Nitric oxide (NO) exerts conflicting effect on tumor growth and progression, depending on its concentration. We aimed to characterize the anti-cancer activity of a new NO donor, Ni(SalPipNONO) belonging to the class of metal-nonoates, in epithelial derived tumor cells, finally exploring its antiangiogenic properties. Tumor epithelial cells were screened to evaluate the cytotoxic effect of Ni(SalPipNONO), which was able to inhibit cell proliferation in a dose dependent manner, being more effective than the commercial DETA/NO. The human lung carcinoma cells A549 were chosen as model to study the anti-cancer mechanisms exerted by the compound. In these cells, Ni(SalPipNONO) inhibited clonogenicity and cell invasion, while promoting apoptosis. The antitumor activity was partly due to NO-cGMP dependent pathway, contributing to reduced cell number and apoptosis, and partly to the salicylaldehyde moiety and reactive oxygen species (ROS) activated ERK1/2 signaling converging on p53 dependent caspase-3 cleavage. An additional contribution by downstream cycloxygenase-2 (COX-2) derived cyclopentenones may explain the tumor inhibitory activities. As NO has been described to affect tumor angiogenesis, we checked this activity both on tumor and endothelial cell co-cultures and in Matrigel in vivo assay. Our data document that Ni(SalPipNONO) was able to both reduce angiogenic factor expression by tumor cells acting on hypoxia inducible factor-1α (HIF-1 α) level, and endothelial cell functions related to angiogenesis. Collectively, these data confirm the potential use of NO donors and in particular Ni(SalPipNONO) acting through multiple mechanisms, as an agent to be further developed to be used alone or in combination with conventional therapy

Recognition and Sensing of Nucleoside Monophosphates by a Dicopper(II) Cryptate

The dicopper complex of a bis-tren cryptand in which the spacer consists of two furane subunits connected in 2,2' by a -CH2- fragment selectively recognizes guanosine monophosphate with respect to other nucleoside monophospates (NMPs) in a MeOH/water solution at pH 7. Recognition is efficiently signaled through the displacement of the indicator 6-carboxyfluorescein bound to the receptor, monitoring its yellow fluorescent emission. Titration experiments evidenced the occurrence of several simultaneous equilibria involving 1:1 and 2:1 receptor/NMP and receptor/indicator complexes. It was demonstrated that the added NMP displaces the indicator from the 2:1 receptor/indicator complex, forming the 1:1 receptor/ analyte inclusion complex. Recognition selectivity is thus ascribed to the nature of nucleotide donor atoms involved in the coordination and their ability to encompass the CuII-CuII distance within the cryptate

Analysis of the Secondary Structure of the Catalytic Domain of Mouse Ras Exchange Factor CDC25Mm

The minimal active domain GEF domain. of the mouse Ras exchange factor CDC25Mm was purified to homogeneity from recombinant Escherichia coli culture. The 256 amino acids polypeptide shows high activity in vitro and forms a stable complex with H-ras p21 in absence of guanine nucleotides. Circular dichroism CD. spectra in the far UV region indicate that this domain is highly structured with a high content of a-helix 42%.. Near UV CD spectra evidenced good signal due to phenylalanine and tyrosine while a poor contribution was elicited by the three tryptophan residues contained in this domain. The tryptophan fluorescence signal was scarcely affected by denaturation of the protein or by formation of the binary complex with H-ras p21, suggesting that the Trp residues, which are well conserved in the GEF domain of several Ras-exchange factors, were exposed to the surface of the protein and they are not most probably directly involved in the interaction with Ras proteins. q1998 Elsevier Science B.

Suggestive criteria for pulmonary tuberculosis in developing countries

Abstract Tuberculosis (TB) represents a major problem in developing countries. Diagnosis is often difficult and mainly relies on clinical criteria and simple laboratory examinations, as cultural methods and molecular biology are not available in most health facilities. In order to evaluate the reliability of clinical criteria to suggest pulmonary TB, a prospective survey was conducted in Wolisso Hospital, South-West Shewa region, Ethiopia. During the period from April 2006 to September 2008, data from 117 consecutive patients from which the diagnosis of TB was made by either positive sputum examination or by typical chest X-ray were examined. The objective was to identify simple and reproducible clinical and laboratory criteria related to pulmonary TB in low-resource health facilities. Patients' symptoms strongly suggesting pulmonary TB were found to be long-lasting cough (>1 month), dyspnoea, chest pain, weight loss, fever, weakness and night sweats; typical TB patients' physical examination showed emaciated condition, with low systolic blood pressure (BP) and low body mass index (BMI); simple laboratory examinations suggestive of TB were high erythrocyte sedimentation rate (ESR) and normocytic anaemia. Absence of both known TB contact and bloody sputum was not significant to rule out TB, and total and differential white blood cells (WBC) count did not help in the diagnosis

The Nitric Oxide Donor [Zn(PipNONO)Cl] Exhibits Antitumor Activity through Inhibition of Epithelial and Endothelial Mesenchymal Transitions

Simple Summary Nitric oxide (NO) plays a critical pathophysiological role in cancer by modulating several processes, such as angiogenesis, tumor growth, and metastatic potential. The aim of this study was to characterize the antitumor effects of a novel NO donor, [Zn(PipNONO)Cl], on the processes of epithelial- and endothelial-mesenchymal transitions (EMT and EndMT), known to actively participate in cancer progression. Two tumor cells lines were used in this study: human lung cancer cells (A549) and melanoma cells (A375), alone and co-cultured with human endothelial cells. Our results demonstrate that both tumor and endothelial cells were targets of NO action, which impaired EMT and EndMT functional and molecular features. Further studies are needed to finalize the therapeutic use of the novel NO donor. Exogenous nitric oxide appears a promising therapeutic approach to control cancer progression. Previously, a nickel-based nonoate, [Ni(SalPipNONO)], inhibited lung cancer cells, along with impairment of angiogenesis. The Zn(II) containing derivatives [Zn(PipNONO)Cl] exhibited a protective effect on vascular endothelium. Here, we have evaluated the antitumor properties of [Zn(PipNONO)Cl] in human lung cancer (A549) and melanoma (A375) cells. Metastasis initiates with the epithelial-mesenchymal transition (EMT) process, consisting of the acquisition of invasive and migratory properties by tumor cells. At not cytotoxic levels, the nonoate significantly impaired A549 and A375 EMT induced by transforming growth factor-beta 1 (TGF-beta 1). Reduction of the mesenchymal marker vimentin, upregulated by TGF-beta 1, and restoration of the epithelial marker E-cadherin, reduced by TGF-beta 1, were detected in both tumor cell lines in the presence of Zn-nonoate. Further, the endothelial-mesenchymal transition achieved in a tumor-endothelial cell co-culture was assessed. Endothelial cells co-cultured with A549 or A375 acquired a mesenchymal phenotype with increased vimentin, alpha smooth muscle actin and Smad2/3, and reduced VE-cadherin. The presence of [Zn(PipNONO)Cl] maintained a typical endothelial phenotype. In conclusion, [Zn(PipNONO)Cl] appears a promising therapeutic tool to control tumor growth and metastasis, by acting on both tumor and endothelial cells, reprogramming the cells toward their physiologic phenotypes

Axial Imidazole Distorsion Effects on the Catalytic and Binding Properties of Chelated Deuterohemin Complexes

The effect of strain in the axial coordination of imidazole to the heme has been studied in the chelate complexes deuterohemin-histidine (DH-His) and deuterohemin-alanylhistidine (DH-AlaHis). Molecular mechanics calculations indicate that three types of distortion of the axial ligand occur in DH-His, due to the relatively short length of the arm carrying the donor group: tilting off-axis, tipping, and inclination of the imidazole plane with respect to the axial Fe-N bond. The effects of tilting (¢ç 10°) and inclination of the imidazole ring (¢ä 17°) are dominant, while tipping is small and is probably of little importance here. By contrast, the axial imidazole coordination is normal in DH-AlaHis and other computed deuterohemin-dipeptide or -tripeptide complexes where histidine is the terminal residue, the only exception being DH-ProHis, where the rigidity of the proline ring reduces the flexibility of the chelating arm. The distortion in the axial iron-imidazole bond in DH-His has profound and negative influence on the binding and catalytic properties of this complex compared to DH-AlaHis. The former complex binds more weakly carbon monoxide, in its reduced form, and imidazole, in its oxidized form, than the latter. The catalytic efficiency in peroxidative oxidations is also reduced in DH-His with respect to DH-AlaHis. The activity of the latter complex is similar to that of microperoxidase-11, the peptide fragment incorporating the heme that results from hydrolytic cleavage of cytochrome

Formation of reactive nitrogen species at biologic heme centers: a potential mechanism of nitric oxide-dependent toxicity.

The peroxidase-catalyzed nitration of tyrosine derivatives by nitrite and hydrogen peroxide has been studied in detail using the enzymes lactoperoxidase (LPO) from bovine milk and horseradish peroxidase (HRP). The results indicate the existence of two competing pathways, in which the nitrating species is either nitrogen dioxide or peroxynitrite. The first pathway involves one-electron oxidation of nitrite by the classical peroxidase intermediates compound I and compound II, whereas in the second pathway peroxynitrite is generated by reaction between enzyme-bound nitrite and hydrogen peroxide. The two mechanisms can be simultaneously operative, and their relative importance depends on the reagent concentrations. With HRP the peroxynitrite pathway contributes significantly only at relatively high nitrite concentrations, but for LPO this represents the main pathway even at relatively low (pathophysiological) nitrite concentrations and explains the high efficiency of the enzyme in the nitration. Myoglobin and hemoglobin are also active in the nitration of phenolic compounds, albeit with lower efficiency compared with peroxidases. In the case of myoglobin, endogenous nitration of the protein has been shown to occur in the absence of substrate. The main nitration site is the heme, but a small fraction of nitrated Tyr146 residue has been identified upon proteolytic digestion and high-performance liquid chromatography/mass spectrometry analysis of the peptide fragments. Preliminary investigation of the nitration of tryptophan derivatives by the peroxidase/nitrite/hydrogen peroxide systems shows that a complex pattern of isomeric nitration products is produced, and this pattern varies with nitrite concentration. Comparative experiments using chemical nitrating agents indicate that at low nitrite concentrations, the enzymatic nitration produces a regioisomeric mixture of nitrotryptophanyl derivatives resembling that obtained using nitrogen dioxide, whereas at high nitrite concentrations the product pattern resembles that obtained using peroxynitrite

Chloride-binding in organic–water mixtures: the powerful synergy of C–H donor groups within a bowl-shaped cavity

2,3,4,5-Tetrafluorobenzyl and imidazolium groups within an open-chain receptor allow for the effective binding of chloride in organic–water solution