Oxidative profile in patients with colon cancer: effects of Ruta chalepensis L.

AIM: To verify the involvement of free radicals in tumor progression and to investigate the effects of an ethanolic extract of Ruta Chalepensis L. and of rutin in blood of patients with colon cancer. MATERIALS AND METHODS: Leaves of Ruta Chalepensis L. were collected in the area around Catania (Italy). For the preparation of the ethanol extract of leaves, an exhaustive extraction of 100 g of the drug was carried out in Soxhlet with 800 ml of 95% ethanol. Fifty-six patients with colorectal cancer were randomly selected for this study; among these, 34 were affected by an early stage (T1 N0 M0 according to scale), while 22 were affected by an advanced stage (T4, N1-2, M0) of cancer. Data obtained from these patients were compared with those of a control group consisting of 20 healthy subjects. Plasma of each sample was used for determining non-proteic antioxidant capacity, thiol groups, lipid hydroperoxides and nitrite/nitrate levels, evaluated by spectrophotometric tests. In addition, percentage of haemolysis was evaluated incubating (for 2 hours at 37 degrees C) erythrocyte suspension with a free radical donor (50 mM 2,2'-azobis-amidino propane chloridrate), in the presence or absence of ethanolic extract of Ruta Chalepensis L. (250 microg/ml) or rutin (1 mM). RESULTS: Non-proteic antioxidant capacity was significantly lower in cancerous patients than in healthy subjects (p < 0.001). This decrease was stage-related. In fact, non-proteic antioxidant capacity resulted lower in advanced than in early colorectal cancer (p < 0.001). The same significant stage-related decrease was observed in plasma thiol groups (p < 0.001). Coherently with the decrease in non-proteic antioxidant capacity and thiol groups, higher levels of lipid hydroperoxides and nitrite/nitrate were observed in patients with colorectal cancer with respect to healthy subjects (p < 0.001) and the increase in these markers of oxidative stress was related to the cancer stadiation. Neoplastic patients also showed an increased percentage of oxidative hemolysis respect to controls and the haemolytic damage was correlated with the stage of colon cancer. Both the extract of Ruta Chalepensis L. and rutin were able to protect erythrocytes from oxidative stress induced by the free radical donor, but the extract of Ruta Chalepensis L. was more effective than rutin. This protective effect was significant only in erythrocytes from patients with early colorectal group, whereas no significant modification was induced by Ruta Chalepensis L. or rutin in red blood cells from advanced colorectal cancer patients exposed to the same experimental conditions. CONCLUSION: Oxidative stress correlates with colon cancer stadiation and both the extract of Ruta chalepensis and rutin are able to protect red blood cells from radical-induced damage. However, their effects are significant in early stages of cancer. So these natural antioxidants might be usefull to prevent carcinogenesis and/or tumor progression.

A fully automated method for accurate measurement of geometrical distortion in magnetic resonance imaging of a 3D-lattice phantom.

This paper describes an automated method for extracting the apparent positions of fiducial points from 2D or 3D images of a phantom. We consider a 3D-lattice phantom for two main reasons: first, ease of manufacture and isotropy of its structure with respect to coordinate projections; second, a connected structure allowing to uniquely assess the adjacency relationship between fiducial points even if geometric distortions arising from main magnet inhomogeneity and gradient fields non-linearity is severe as observed in open-bore systems. In order to validate our proposed method and compare different choices for the parameters of our phantom (i.e. number and distance between grids and thickness of its branches) we developed in-house a software for simulating 2D or 3D volume images of the phantom, using customizable MRI sequence parameters and Spherical Harmonic Coefficients for the fields. We deem worthy of note that using simulated images is the only way to evaluate the estimated position error, since it allows to compare the estimates to their theoretical counterparts. Furthermore, the use of simulated images allows to evaluate the robustness of the method with respect to image quality in terms of Signal-to-Noise Ratios and geometric distortion, and allows to evaluate different phantom geometries without having to manufacture them. The proposed method can be easily extended to phantoms having an arbitrary overall shape, as long as it is a fully connected structure. Specifically, it is easy to design a phantom with fiducial points laying outside of the homogeneity sphere, so that indirect measurement of the fields becomes possible, for example by using the recent method proposed by Acquaviva et al. To the best of our knowledge, the proposed method outperforms other state-of-the-art methods, with an average positioning offset of 0.052 mm (with a 0.99 quantile of 0.12 mm) when working on images featuring a differential Signal-to-Noise Ratio within Region-of-Interest (ROI) equal to 105 (20.2 dB) and a ROI-to-background SNR of 20 dB. Estimating the positions of 6859 fiducial points in a volume, our highest density case, was carried out in less than 30 min on a desktop personal computer.

Blond and blood juice supplementation in high fat diet fed mice: effect on antioxidant status and DDAH/ADMA pathway.

Non-alcoholic fatty liver disease (NAFLD) is the most common liver disease spread throughout the world. The most frequent causes of death in NAFLD patients are due both to liver and cardiovascular damage. Several pathways, including the dimethylarginine dimethylaminohydrolase (DDAH)/asymmetric dimethylarginine (ADMA) pathway, are involved in the pathogenesis of NAFLD. It has been reported that ADMA plasmatic levels are increased in patients with hepatic dysfunction such as NAFLD. Although many studies demonstrated that some foods are effective in the treatment of NAFLD, few studies have evaluated their effects with respect to the prevention of the disease. It has been reported that sweet orange juice (OJ) consumption may be associated with potential health benefits. However, some varieties of sweet orange are more effective than others. The aim of the present paper was to investigate the effect of blond and blood sweet orange juice in prevention of NAFLD by evaluating its ability to improve liver steatosis in mice with diet-induced obesity, reducing oxidative stress and affecting the DDAH/ADMA pathway. Results obtained in our experimental conditions evidenced that blood orange juice rather than blond orange juice was more effective. Blood orange juice or blond orange juice enriched in anthocyanins may represent a promising dietary option for the prevention of fatty liver disease.

Evaluation of cell tolerability of a series of lipoamino acids using biological membranes and a biomembrane model.

The interaction of a series of amphiphilic 2-alkyl aminoacids (lipoamino acids, LAAs) with different cell cultures and biomembrane models was investigated. LAAs can be useful promoieties to modify the physico-chemical properties of many drugs, and in particular their lipophilicity. Tests were performed in vitro on mammalian cells (murine astrocytes) and human red blood cells (haemolysis), and in vivo on rabbit eye as alternative models to assess the tolerability or the potential damaging effects of these compounds on different biological systems. The mode of interaction of LAAs with pure phospholipid multilamellar liposomes, taken as a biomembrane model, was also analysed by differential scanning calorimetry experiments. Different tolerability/toxicity patterns were obtained in the various models; in particular, the most lipophilic terms of the series, methyl 2-aminohexadecanoate (LAA16), displayed haemolytic activity and toxicity for mouse astrocyte cultures. A specific assay confirmed that LAA16 acted at level of cell membranes, while neither any damaging effects on nucleus or apoptotic induction were observed. The shorter-chain LAAs and the tetradecyl homologue (LAA14) showed the best compatibility with the various cell models.

Pharmacological induction of heme oxygenase-1 inhibits iNOS and oxidative stress in renal ischemia-reperfusion injury.

Nitric oxide (NO), produced by nitric oxide synthase, is implicated in the pathophysiology of renal ischemia/reperfusion (I/R) injury. This study sought to elucidate the impact of pharmacological induction of heme oxygenase-1 (HO-1) on renal I/R injury. Rats were subjected to 45 minutes of renal ischemia followed by various times of reperfusion (30 minutes, 1 hour, or 3 hours). Plasma from sacrificed rats was obtained, and the kidneys processed for the expression of iNOS, cleaved caspase-3, p38MAPK and for immunohistochemical analysis. Furthermore, we determined renal and plasma levels of lipid hydroperoxides, total thiol groups, and plasmatic NO2-/NO3- formation. Our results showed a time-dependent increase in iNOS expression, which was also confirmed by increased plasma formation of NO2-/NO3-. Interestingly, this effect was reversed by pretreatment (12 hours) with SnCl2, a potent and specific inducer of renal HO-1 expression and activity, or by intraperitoneal injection of biliverdin (10 mg/kg). Furthermore, we observed a concomitant reduction in plasma and renal LOOH formation, a normalization of renal total thiol content, a reduction of caspase-3-mediated apoptosis, and a significant increase in p38MAPK phosphoration. Taken together, these results suggested that HO-1 and its byproduct biliverdin play major roles in the pathophysiological cascade leading to renal I/R injury.

Moringa oleifera Lam. improves lipid metabolism during adipogenic differentiation of human stem cells.

OBJECTIVE: Moringa oleifera Lam., a multipurpose tree, is used traditionally for its nutritional and medicinal properties. It has been used for the treatment of a variety of conditions, including inflammation, cancer and metabolic disorders. MATERIALS AND METHODS: We investigated the effect of Moringa oleifera Lam. on adipogenic differentiation of human adipose-derived mesenchymal stem cells and its impact on lipid metabolism and cellular antioxidant systems. RESULTS: We showed that Moringa oleifera Lam. treatment during adipogenic differentiation reduces inflammation, lipid accumulation and induces thermogenesis by activation of uncoupling protein 1 (UCP1), sirtuin 1 (SIRT1), peroxisome proliferator-activated receptor alpha (PPARα), and coactivator 1 alpha (PGC1α). In addition, Moringa oleifera Lam. induces heme oxygenase-1 (HO-1), a well established protective and antioxidant enzyme. Finally Moringa oleifera Lam. significantly decreases the expression of molecules involved in adipogenesis and upregulates the expression of mediators involved in thermogenesis and lipid metabolism. CONCLUSIONS: Our results suggest that Moringa oleifera Lam. may promote the brown remodeling of white adipose tissue inducing thermogenesis and improving metabolic homeostasis.

Comparative stability analysis of the thyroid transcription factor 1 and Antennapedia homeodomains: evidence for residue 54 in controlling the structural stability of the recognition helix.

The thyroid transcription factor 1 homeodomain (TTF-1 HD) shows a peculiar DNA-binding specificity which is partially dictated by several amino acids of the recognition helix. TTF-1 preferentially recognizes sequences containing the 5'-CAAG-3' core motif while most other homeodomains, such as Antennapedia (Antp), recognizes sites containing the 5'-TAAT-3' core motif. Since phenomena of 'induced fit' may occur during protein/DNA interaction, a primary role for high affinity binding and target discrimination has to be searched in the effect played by subtle structural determinants in these proteins. By using spectroscopic analysis in aqueous solution, we compared the structural stability of TTF-1 and Antp homeodomains. Although the three-dimensional structural architecture of homeodomains is conserved, some differences are detectable in terms of their structural stability. At 24 degrees C the TTF-1 HD is less structured than the Antp HD with 24 and 34% of the residues in the alpha-helical conformation, respectively. This poor folded structure reflects into different thermal and isothermal stability between the two homeodomains. TTF-1 HD exhibits a Tm of 39 degrees C and is stabilized by a delta GDH2O of +1487 cal/mol, calculated by Urea unfolding, while Antp HD exhibits a Tm of 48 degrees C and is stabilized by a delta GDH2O of +2742 cal/mol. By using mutants of both TTF-1 and Antp HDs we demonstrate that one of the major determinants in controlling the structural stability of the recognition helix is the residue at position 54. Since previous studies have shown that also residue at position 56 is involved in stabilization of the recognition helix, we conclude that the structure of this critical element is controlled by an interplay between residues at position 54 and 56 of the homeodomain.

Thyroglobulin binding and TSH regulation of the RHL-1 subunit of the asialoglycoprotein receptor in rat thyroid.

The ability of asialo-thyroglobulin to bind the thyroid RHL-1 subunit of the asialoglycoprotein receptor has been investigated. Ligand blot assays show that the recombinant carbohydrate recognition domain of the thyroid RHL-1 subunit specifically interacts with rat desialated thyroglobulin. Moreover, RT-PCR and Western blot assays show that TSH deprivation decreases RHL-1 expression in PC C13 thyroid differentiated cells whereas insulin deprivation does not have any effect. The simultaneous absence of both TSH and insulin dramatically decreases the level of RHL-1 expression.

Calcium interaction with bovine thyroglobulin: stoichiometry and structural consequences of calcium binding.

Gel filtration studies show that the thyroglobulin (Tg) molecule (dimer) binds from 18 to 50 Ca2+ ions. At pH 7.5 Tg binds 18 Ca2+ ions with a Kd of 1.3 x 10(-5) M, and 50 Ca2+ ions with a Kd of 5.5 x 10(-4) M. The binding of calcium to bovine thyroglobulin increases the absorption band of iodoamino acid residues at 315 nm. In the presence of Ca2+, the fluorescence intensity of 1-anilino-8-naphthalene sulfonate (ANS) is increased about 5-fold by Tg, with a shift in the fluorescence emission maximum from 505 to 490 nm. Thus, thyroglobulin possesses two classes of calcium binding sites with different affinities. The data reported indicate, also, that Ca2+ binding to Tg increases the hydrophobicity of the surface of the molecule.

Antibacterial activity of Helleborus bocconei Ten. subsp. siculus root extracts.

AIM OF THE STUDY: Roots of Helleborus bocconei Ten. subsp. siculus (Schiffner) Merxm. & Podl. are widely used in veterinary folk medicine in Sicily (Italy) to diagnose and cure lower respiratory tract infections in cattle. This study intended to evaluate the in vitro antibacterial activity of the methanolic root extract of Helleborus bocconei Ten. subsp. siculus, and of the bufadienolide and ecdysteroid fractions extracted from its roots, against strains belonging to species commonly associated with respiratory tract infections. MATERIALS AND METHODS: The phytochemical screening of the previously prepared plant extracts was carried out by chemical, thin-layer chromatography and spectroscopic methods. The in vitro antibacterial activity of the extracts against seven different standard bacterial strains was evaluated by broth microdilution. RESULTS: According to the present study, Helleborus bocconei Ten. subsp. siculus roots contain bufadienolides and ecdysteroids, and the extracts containing these compounds, as well as the crude methanolic root extract, show antibacterial activity against microorganisms responsible for respiratory infections. In particular, the bufadienolide extract has the highest inhibitory activity against all the tested organisms, and, as the other extracts, shows the lowest MIC values (100mug/ml) against Moraxella catarrhalis and Streptococcus pneumoniae. CONCLUSIONS: Since the roots of Helleborus bocconei Ten. subsp. siculus contain substances with antibacterial activity, the traditional use of this plant may also derive from its antibacterial properties.

Imidazole derivatives as antioxidants and selective inhibitors of nNOS.

The reperfusion of ischemic tissue often delays its physiological and functional recovery; this paradoxical effect is ascribed to increased release of free radicals including O(2)(-) and NO. For these reasons, scavenging reactive oxygen species or inhibition the NO synthesis has been shown to result in an enhanced neuronal survival after cerebral ischemia. Many authors believe that therapy for stroke patients would be a cocktail of drugs with various mechanisms of action. Combination therapy is a difficult and complicated avenue for drug development because of the possibility of drug-drug interactions. An alternative approach would be to combine multiple activities within the same compound. In consideration of the free-radical scavenging and inhibitory effect on NOS of various natural and synthetic compounds, the aim of this study was to analyze the antioxidant properties of some imidazole derivatives previously synthesized in our laboratory. Results obtained in the present study provide evidence that tested compounds exhibit interesting antioxidant properties, expressed either by their capacity to scavenge free radicals or their ability to reduce lipid peroxidation. In particular, compounds A and B represent chemical structures which can be easily modified to improve the observed antioxidant properties and to provide new therapeutic strategies focused on multiple downstream events.

Antioxidant activity and protective effect on DNA cleavage of extracts from Cistus incanus L. and Cistus monspeliensis L.

The genus Cistus includes many typical species of Mediterranean flora; Cistus species are used as antidiarrhetics, as general remedies for treatment of various skin diseases in folk medicine and as anti-inflammatory agents. These species contain flavonoids that are considered to be chain-breaking antioxidants. In this work, we have investigated the effects of crude aqueous extracts from Cistus incanus and Cistus monspeliensis on DNA cleavage and their free-radical scavenging capacity. In addition, their effect on lipid peroxidation in rat liver microsomes was evaluated. These extracts showed a protective effect on DNA cleavage and a dose-dependent free-radical scavenging capacity; Cistus monspeliensis was more active than Cistus incanus; these results were confirmed by a significant inhibition of lipid peroxidation in rat liver microsomes. The experimental evidence, therefore, suggests that because of their antioxidant activity these extracts may offer excellent photoprotection for skin and may be useful in the treatment of human diseases where oxidative stress plays a key role.

Enhanced resistance of adriamycin-treated MCR-5 lung fibroblasts by increased intracellular glutathione peroxidase and extracellular antioxidants.

Considerable evidence indicates that reactive oxygen species play an etiological role in both cardiotoxicity and the skin necrosis induced by adriamycin (ADM). An increase in glutathione peroxidase activity on addition of selenium to cultured MCR-5 lung fibroblasts was observed; this increase was accompanied by enhanced cellular resistance to ADM toxicity. Moreover, the presence of exogenous antioxidant systems, such as superoxide dismutase, catalase, vitamin E, dimethylsulfoxide, and desferroxamine, an iron chelating agent, resulted in significant protection from ADM-mediated damage.

Calcium-induced changes in thyroglobulin conformation.

Polyethylene glycol solutions (10% w/v) were used to detect the effect of mono- and divalent cations on some properties of thyroglobulin. It is shown that in presence of 10% w/v polyethylene glycol in 0.01 M Tris-HCl, pH 7.5, calcium (less than 0.05 M) modifies the solubility, the sedimentation rate, and the Stokes' radius of thyroglobulin, while monovalent cations up to 0.6 M do not effect any of these properties. These findings can be explained by an increase in molecular compactness of thyroglobulin. Furthermore, it was shown that a synthetic polymer, polyethylene glycol, could be used to detect conformational changes.

Topology of the thyroid transcription factor 1 homeodomain-DNA complex.

The topology of the thyroid transcription factor 1 homeodomain (TTF-1HD)-DNA complex was investigated by a strategy which combines limited proteolysis and selective chemical modification experiments with mass spectrometry methodologies. When limited proteolysis digestions were carried out with the protein in the absence or presence of its target oligonucleotide, differential peptide maps were obtained from which the amino acid residues involved in the interaction could be inferred. Similarly, selective acetylation of lysine residues in both the isolated and the complexed homeodomain allowed us to identify the amino acids protected by the interaction with DNA. Surface topology analysis of isolated TTF-1HD performed at neutral pH was in good agreement with the three-dimensional structure of the molecule as determined by NMR studies under acidic conditions. Minor differences were detected in the C-terminal region of the protein which, contrary to NMR data, showed no accessibility to proteases. Analysis of the complex provided an experimental validation of the model proposed on the basis of the homology with the homeodomain structures described so far. An increased accessibility of the C-terminal region was observed following the interaction, suggesting its displacement from the protein core by the oligonucleotide molecule. Comparative experiments with DNA fragments differing in sequence and binding capabilities highlighted structural differences among the complexes, mainly located in the N-terminal region of the homeodomain, thus accounting for their different dissociation constants.

Prediction of the secondary structure of the carboxy-terminal third of rat thyroglobulin.

A secondary structure prediction has been made using the available primary sequence data of the proposed carboxy-terminal of rat thyroglobulin. The model predicts 22% alfa-helix, 28% beta-structure and 17% beta turns. Out of the 8 possible carbohydrate acceptor-sites (Asn-x-Ser/Thr), 3 (residues 136, 368, 782) are associated with peptide sequences which favour the formation of beta-turn or loop-structures and are located in high hydrophilic regions. The entire sequence is predicted to be made up of two domains: one of them is highly structured, contains the hormonogenic sites, a cluster of tyrosines and at least one carbohydrate acceptor site.

Presence of dityrosine bridges in thyroglobulin and their relationship with iodination.

The presence of dimeric thyroglobulin (19S), after reduction and alkylation, suggests that within thyroglobulin there may be intermolecular cross-links, other than disulphide bridges. However, the nature of these intermolecular cross-links is still unknown. In this study, we show the presence of 3-3' dityrosine bridges in the molecule of bovine thyroglobulin by NMR and fluorescence studies. Also, we evaluated the role of iodination in dityrosine formation in vivo and in vitro.

Bioflavonoids as antiradicals, antioxidants and DNA cleavage protectors.

Flavonoids have recently aroused considerable interest because of their broad pharmacological activity. In fact, flavonoids have been reported to have antiviral, antiallergic, antiplatelet, anti-inflammatory and antitumoral activities. The pharmacological properties of bioflavonoids have been ascribed both to the concomitant inhibition of enzymes involved in the production of free radicals and to their free-radical scavenging and iron chelating capacity. However the antioxidant capacity of bioflavonoids due to free-radical scavenging and/or to iron chelating is still controversial. In this study, we have investigated the free-radical scavenging capacity of bioflavonoids (rutin, catechin, and naringin). In addition, the effects of these polyphenols on xanthine oxidase activity, spontaneous lipid peroxidation, and DNA cleavage were investigated. The bioflavonoids under examination showed a dose-dependent free-radical scavenging effect, a significant inhibition of xanthine oxidase activity, and an antilipoperoxidative capacity. In addition, they showed a protective effect on DNA cleavage.