Effect of the liposomal form of taxifolin complexes with metals of variable valence on skin regeneration after chemical burn

The effect of the liposomal form formed by taxifolin and metals of variable valency was investigated. It was shown that the application of preparations based on the free flavonoid and its complexes with Fe(II/III) and Cu(II) ions after chemical burns results in a more effective skin regeneration and the repair of hair follicles and sebaceous glands. A tendency for a more effective wound healing after the applications of taxifolin-Cu(II) and taxifolin-Fe(III) liposomal complexes versus control was observed. It was assumed that the mechanism of action of these preparations is based on the oxidative polymerization and conjugation of the flavonoid, which results in the utilization of toxic metabolites and lipid peroxidation products.     

Study of membranotropic and antioxidant activity of flavonoids and their complexes with ferric iron

We studied the interaction with liposomes and the antioxidant activity of flavonoid (quercetin, catechin, taxifolin) complexes with iron(III). It was found that the lipophilicity of complexes depends on an iron:flavonoid ratio and grows at a ratio of 1 to 1, while complexes in a 2: 1 ratio were the most effective to slow down the lipid peroxidation and restore radical 2,2-diphenyl-1-picrylhydrazyl. Thus, the stoichiometry of complexes formed in aqueous solution, may differ from the stoichiometry of complexes that most effectively protect membranes from peroxidation.     

Influence of metal ions on flavonoid protection against asbestos-induced cell injury

Influence of metal ions (Fe2+, Fe3+, Cu2+, Zn2+) on the protective effect of rutin, dihydroquercetin, and green tea epicatechins against in vitro asbestos-induced cell injury was studied. Metals have been found to increase the capacity of rutin and dihydroquercetin to protect peritoneal macrophages against chrysotile asbestos-induced injury. The data presented here show that this effect is due to the formation of flavonoid metal complexes, which turned out to be more effective radical scavengers than uncomplexed flavonoids. At the same time epicatechins and their metal complexes have similar antiradical properties and protective capacities against the asbestos induced injury of macrophages. Metal complexes of all flavonoids were found to be considerably more potent than parent flavonoids in protecting red blood cells against asbestos-induced injury. It was also found that the metal complexes of all flavonoids were absorbed by chrysotile asbestos fibers considerably better than uncomplexed compounds and probably for this reason flavonoid metal complexes have better protective properties against asbestos induced hemolysis. Thus, the results of the present study show that flavonoid metal complexes may be effective therapy for the inflammatory response associated with the inhalation of asbestos fiber. The advantage of their application could be the strong increase in ROS scavenging by flavonoids and finally a better cell protection under the conditions of cellular oxidative stress.     

Complexation of desferricoprogen with trivalent Fe, Al, Ga, In and divalent Fe, Ni, Cu, Zn metal ions: effects of the linking chain structure on the metal binding ability of hydroxamate based siderophores

Complexes of the natural siderophore, desferricoprogen (DFC), with several trivalent and divalent metal ions in aqueous solution were studied by pH-potentiometry, UV-Vis spectrophotometry and cyclic voltammetry. DFC was found to be an effective metal binding ligand, which, in addition to Fe(III), forms complexes of high stability with Ga(III), Al(III), In(III), Cu(II), Ni(II) and Zn(II). Fe(II), however, is oxidized by DFC under anaerobic conditions and Fe(III) complexes are formed. By comparing the results with those of desferrioxamine B (DFB), it can be concluded that the conjugated beta-double bond slightly increases the stability of the hydroxamate chelates, consequently increases the stability of mono-chelated complexes of DFC. Any steric effect by the connecting chains arises only in the bis- and tris-chelated complexes. With metal ions possessing a relatively big ionic radius (Cu(II), Ni(II), Zn(II), In(III)) DFC, containing a bit longer chains than DFB, forms slightly more stable complexes. With smaller metal ions the trend is the opposite. Also a notable difference is that stable trinuclear complex, [Cu(3)L(2)], is formed with DFC but not with DFB. Possible bio-relevance of the Fe(II)/Fe(III) results is also discussed in the paper.     

Column chromatography of human small-intestinal maltase, isomaltase and invertase activities

1. The maltase, isomaltase and invertase (sucrase) activities of solubilized mucosal preparations from human jejunum and ileum were studied with column chromatography on anion-exchange (diethylaminoethyl- and triethylaminoethyl-)cellulose and Sephadex G-200 gel. 2. On ion-exchange cellulose columns both kinds of enzyme preparations yielded two major disaccharidase peaks. The first peak contained maltase Ia (=isomaltase) and maltase Ib (=invertase). The second peak contained maltase II and maltase III. 3. On Sephadex G-200 gel columns jejunal preparations yielded the corresponding peaks as on ion-exchange columns, but the peaks appeared in the reverse order in the effluent. The ileal preparation studied yielded a single peak on gel columns, containing all the activities studied and eluted with the `void volume'. 4. Precipitation with ethanol did not affect the behaviour of the enzymes during ion-exchange chromatography. When gel filtration was performed after ethanol precipitation of the enzymes, however, two peaks were obtained also with the ileal preparation, and subfractionation of the invertase was obtained with both kinds of preparations. 5. The second peak from ion-exchange chromatograms, containing maltase II and maltase III, on concentration was found to have very weak isomaltase activity, probably exerted by these enzymes as such. This activity accounts for only about 1% of the total isomaltase activity of the mucosa. 6. The results support the concept of the specificity of the human small-intestinal disaccharidases previously described after heat-inactivation experiments. The subfractionation of the invertase that under certain conditions is seen on Sephadex G-200 columns appears most likely to be an artifact. Consequently the nomenclature for the human maltose-, isomaltose- and sucrose-splitting enzymes proposed by another research group after gel-filtration chromatography studies should be abandoned. It seems more logical to keep the nomenclature based on heat inactivation [maltase Ia (=isomaltase), maltase Ib (=invertase or sucrase), maltase II and maltase III] until increased knowledge about the specificity and structure of these enzymes makes possible a more rational nomenclature.     

Liposomal form of dihydroquercetin contributes to skin regeneration after thermal burns

It was found that dihydroquercetin, a flavonoid of plant origin, localized in lecitin nanoparticles with glycine amino acid, reduced inflammatory reactions in wound zones after thermal burns. The application of the liposomal complex to burn trauma stabilized the endogenous antioxidant system and minimized the area of secondary necrosis in the wound. The intensification of skin regeneration and repair of hair follicles and sebaceous glands were also observed.     

Temozolomide,quercetin and cell death in the MOGGCCM astrocytoma cell line

The aim of the present study was to investigate the effect of Temozolomide (an alkylating chemotherapeutic agent) and quercetin (natural flavonoid) on cell death in the human astrocytoma cell line MOGGCCM (WHO grade III). Our results indicate that Temozolomide induces autophagy, while quercetin promotes severe necrosis in the cell line in a manner dependent on the drug concentration. We demonstrated for the first time that combinations of both drugs were much more effective in programmed cell death induction in glioma cells. At a low (5 μM) drug concentration, quercetin potentiated a pro-autophagic effect of Temozolomide, while after treatment with a higher drug concentration (30 μM), autophagy switched to apoptosis. Temozolomide attenuated the toxic effect of quercetin. Apoptosis was mediated by the mitochondrial pathway and the activation of caspase 3 and cytochrome C release, but no changes in caspase 8 expression was observed. It was accompanied by decreased mitochondrial membrane potential and inhibition of Hsp27 and Hsp72 expression. Autophagy was correlated with an increased level of LC3II. Temozolomide and quercetin also inhibited migratory phenotype of MOGGCCM cells and changed the nuclei morphology from a circular to an irregular shape. Our results indicate that quercetin acts in synergy with Temozolomide and when used in combination rather than in separate pharmacological application, both drugs are more effective in programmed cell death induction. Temozolomide administered with quercetin seems to be a potent and promising combination which might be useful in glioma therapy.     

Physicochemical study of floranol, its copper(II) and iron(III) complexes, and their inhibitory effect on LDL oxidation

The antioxidant activity of floranol (3,5,7,2'-tetrahydroxy-6-methoxy-8-prenylflavanone), a new flavonoid isolated from the roots of Dioclea grandiflora, was evaluated by the inhibition of human low-density lipoprotein (LDL) oxidation. Floranol increased its oxidation lag-phase significantly in a dose-dependent manner. As the antioxidant mechanism may involve metal coordination, we have undertaken a detailed study of floranol interactions with Cu(II) and Fe(III) by combination of UV-visible (UV-Vis) and mass spectrometries and cyclic voltammetry. The acidity constants of the ligand as well as the stability constants of the metal complexes were calculated. The pKa values of 6.58, 11.97 and 13.87 were determined and the following acidity order is proposed 7-OH>5-OH>2'-OH. The best fit between experimental and calculated spectra was obtained assuming the formation of two Cu(II) complexes: [CuL] logbeta=19.34+/-0.05 and [CuL(2)](2-) logbeta=26.4+/-0.10 and three Fe(III) complexes: [FeL(3)](3-) logbeta=44.72+/-0.09, [FeL(2)](-) logbeta=35.32+/-0.08 and [FeL](+) logbeta=19.51+/-0.04. In addition, copper and iron reduction is less favorable in the presence of floranol. These results indicate that floranol can efficiently bind Cu(II) and Fe(III) ions thus preventing their effect on LDL oxidation.     

New Effective Method for Analysis of the Component Composition of Enzyme Complexes from <Emphasis Type="Italic">Trichoderma reesei</Emphasis>

A method for analysis of the component composition of multienzyme complexes secreted by the filamentous fungus Trichoderma reesei was developed. The method is based on chromatofocusing followed by further identification of protein fractions according to their substrate specificity and molecular characteristics of the proteins. The method allows identifying practically all known cellulases and hemicellulases of T. reesei: endoglucanase I (EG I), EG II, EG III, cellobiohydrolase I (CBH I), CBH II, xylanase I (XYL I), XYL II, beta-xylosidase, alpha-L-arabinofuranosidase, acetyl xylan esterase, mannanase, alpha-galactosidase, xyloglucanase, polygalacturonase, and exo-beta-1,3-glucosidase. The component composition of several laboratory and commercial T. reesei preparations was studied and the content of the individual enzymes in these preparations was quantified. The influence of fermentation conditions on the component composition of secreted enzyme complexes was revealed. The characteristic features of enzyme preparations obtained in "cellulase" and "xylanase" fermentation conditions are shown.     

The ratio of oxidative phosphorylation complexes I-V in bovine heart mitochondria and the composition of respiratory chain supercomplexes

The ratios of the oxidative phosphorylation complexes NADH:ubiquinone reductase (complex I), succinate:ubiquinone reductase (complex II), ubiquinol:cytochrome c reductase (complex III), cytochrome c oxidase (complex IV), and F1F0-ATP synthase (complex V) from bovine heart mitochondria were determined by applying three novel and independent approaches that gave consistent results: 1) a spectrophotometric-enzymatic assay making use of differential solubilization of complexes II and III and parallel assays of spectra and catalytic activities in the samples before and after ultracentrifugation were used for the determination of the ratios of complexes II, III, and IV; 2) an electrophoretic-densitometric approach using two-dimensional electrophoresis (blue native-polyacrylamide gel electrophoresis and SDS-polyacrylamide gel electrophoresis) and Coomassie blue-staining indices of subunits of complexes was used for determining the ratios of complexes I, III, IV, and V; and 3) two electrophoretic-densitometric approaches that are independent of the use of staining indices were used for determining the ratio of complexes I and III. For complexes I, II, III, IV, and V in bovine heart mitochondria, a ratio 1.1 +/- 0.2:1.3 +/- 0.1:3:6.7 +/- 0.8:3.5 +/- 0.2 was determined.     

Kinetics and mechanisms of reduction of Cu(II) and Fe(III) complexes by soybean leghemoglobin alpha

The reduction of low-molecular-weight Cu(II) and Fe(III) complexes by soybean leghemoglobin alpha was characterized using both kinetic analysis and 1H-NMR experiments. Whereas Fe(III) (CN)6(3-) was reduced through an outer sphere transfer over the exposed heme edge, all other Cu(II) and Fe(III) complexes investigated were reduced via a site-specific binding of the metal to the protein. Reduction of all metal complexes was enhanced by decreasing pH while only Fe(III)NTA reduction kinetics were altered by changes in ionic strength. Rates of reduction for both Cu(II) and Fe(III) were also affected inversely by the effective binding constant of the metal chelate used. NMR data confirmed that both Cu(II)NTA and Fe(III)NTA were bound to specific sites on the protein. Cu(II) bound preferentially to distal His-61 and Fe(III) exerted its greatest effect on two surface lysine residues with epsilon proton resonances at 3.04 and 3.12 ppm. The Fe(III)NTA complex also had a mild but noticeable line broadening effect on the distal His-61 singlet resonance near 5.3 ppm. Like hemoglobin and myoglobin, leghemoglobin might function not only as an oxygen carrier, but also as a biological reductant for low-molecular-weight Cu(II) and Fe(III) complexes.     

Effect of Organic Acids on Reactions Catalyzed by Manganese Peroxidase from Phanerochaete chrysosporium

The effect of several organic acids on the oxidation of Mn(II) catalyzed by manganese peroxidase was studied. Reactivities of manganese peroxidase and chemically prepared Mn(III) organic acid complexes towards phenolic compounds were compared. If lactate appears to be the best complexant for manganese peroxidase activity, chemically prepared Mn(III)—lactate complex is a less effective oxidant towards phenolic compounds than other Mn(III)—complexes. Our results agree with the hypothesis that certain organic acids are involved in the catalytic cycle of manganese peroxidase. Malonate and lactate seem to be the most attractive complexants for practical applications of manganese peroxidase and were used in enzymatic treatment of hardwood kraft pulp. Bleaching of kraft pulp was studied and after alkaline extraction, a significant decrease of kappa number was measured. The bleaching was enhanced in lactate buffer.     

The influence of chelating agents upon the dissimilatory reduction of Fe(III) by Shewanella putrefaciens

The ability of S. putrefaciens to reduce Fe(III) complexed by a variety of ligands has been investigated. All of the ligands tested caused the cation to be more susceptible to reduction by harvested whole cells than when uncomplexed, although some complexes were more readily reduced than others. Monitoring rates of reduction by a ferrozine assay for Fe(II) formation proved inadequate using Fe(III) ligands giving Fe(II) complexes of low kinetic lability (e.g. EDTA). A more suitable assay for Fe(III) reduction in the presence of such ligands proved to be the observation of associated cytochrome oxidation and re-reduction. Where possible, an assay for Fe(III) reduction based upon the disappearance of Fe(III) complex was also employed. Reduction of all Fe(III) complexes tested was totally inhibited by the presence of O₂, partially inhibited by HQNO and slower in the absence of a physiological electron donor. Upon cell fractionation, Fe(III) reductase activity was detected exclusively in the membranes. Using different physiological electron donors in assays on membranes, relative reduction rates of Fe(III) complexes complemented the data from whole cells. The differences in susceptibility to reduction of the various complexes are discussed, as is evidence for the respiratory nature of the reduction.     

Interaction of Ribonucleotides with Metal Hexacyanocobaltate(III): A Possible Role in Chemical Evolution

It has been proposed that metal cyanide complexes would have acted as effective prebiotic catalysts. Insoluble metal cyanide complexes could have concentrated biomonomers from the dilute prebiotic soup, facilitating certain prebiotic reactions. In the light of the above hypothesis, interaction of four ribonucleotides, namely 5′-AMP, 5′-GMP, 5′-CMP, and 5′-UMP with copper(II)- and cadmium(II) hexacyanocobaltate(III) has been studied. The interaction was found to be maximum at neutral pH. 5′-GMP showed greater interaction with both the metal hexacyanocobaltate(III) while copper(II) hexacyanocobaltate(III) showed greater uptake than cadmium(II) hexacyanocobaltate(III) for all the four ribonucleotides studied. Infrared spectral studies of ribonucleotides, metal hexacyanocobaltate(III) and ribonucleotide – metal hexacyanocobaltate(III) adducts indicated that the nitrogen base and phosphate moiety of ribonucleotides interact with outer divalent metal ion present in the lattice of metal hexacyanocobaltate(III).     

Effect of alpha- and gamma-tocopherols on the cytochrome P-450 system in the liver of rats with E-avitaminosis. Effectiveness of liposomal forms of gamma-tocopherol

The effects of low doses of intraperitoneally injected alpha- and gamma-tocopherols dispersed in isotonic solutions of NaCl and of liposomal gamma-tocopherol on endogenous lipid peroxidation and the cytochrome P-450 system of vitamin E-deficient rats were compared. It was found that all tocopherol preparations under study caused a twofold decrease of the lipid Schiff base concentration in the liver in comparison with tocopherol-deficient rats. However, the increase in cytochrome P-450 and b5 levels and the high functional activity of the microsomal oxidation system in the benz(a)pyrene hydroxylation reaction was induced only after liposomal gamma-tocopherol injection. It was concluded that the efficiency of the liposomal form of gamma-tocopherol can be conditioned both by preventing gamma-tocopherol oxidation in lecithin vesicles during its transport and/or by an increased liposomal gamma-tocopherol uptake by the liver from the circulating blood.     

A sensitive method for staining proteins transferred to nitrocellulose sheets

A simple physical method to determine the monomer concentration of detergents below as well as above the critical micelle concentration based on the bubble-pressure measurement is described. Aggregated surfactant molecules (micelles) and phospholipid vesicles if present in the sample will not disturb the measurements. Three applications of the method relevant to the preparation of liposomes are shown: (i) measurements of critical micelle concentrations, (ii) evaluation of the affinity constant of the interaction of detergents with liposomal membranes, and (iii) monitoring of residual detergent in liposome preparations during dialysis or after gel chromatography of mixed micelle-derived liposomes. It was found that the efficiency of detergents to produce liposomes during their removal depends on their critical micelle concentrations as well as on their affinity to liposomal membranes.     

Spectral characteristics of PS II reaction centres: as isolated preparations and when integral to PS II core complexes

The discovery that the native PS II enzyme undergoes charge separation via an absorption extending to 730 nm has led us to re-examine the low-temperature absorption spectra of Nanba-Satoh PS II reaction centre preparations with particular focus on the long wavelength region. It is shown that these preparations do not exhibit absorption in the 700-730 nm region at 1.7 K. Absorption in the Nanba-Satoh type preparations analogous to the 'red tail' as observed in functional PS II core complexes is likely shifted to higher energy by >20 nm. Spectral changes associated with the stable reduction of pheo(a) in chemically treated reaction centre preparations are also revisited. Dithionite treatment of PS II preparations in the dark leads to changes of pigment-pigment and/or pigment-protein interactions, as evidenced by changes in absorption and CD spectra. Absorption and CD changes associated with stable Pheo(D1) photo-reduction in PS II core complexes and Nanba-Satoh preparations are compared. For Nanba-Satoh preparations, Q(y) bleaches are approximately 3x broader than in PS II core complexes and are blue-shifted by approximately 4 nm. These data are discussed in terms of current models of PS II, and suggest a need to consider protein-induced changes of some electronic properties of reaction centre pigments.     

Unfolding of iron and copper complexes of human lactoferrin and transferrin

1. Human lactoferrin and transferrin are capable of binding two iron or copper ions into specific binding sites in the presence of bicarbonate. 2. Urea and several alkyl ureas have been effective in unfolding these metal-protein complexes. 3.Biphasic transitions are observed for the unfolding of each of the metal complexes of these proteins as determined by direct visible spectroscopy suggesting the release of iron(III) and Cu(II) ions from both of these metal-binding proteins during the unfolding process. 4. Greater stabilization and increased resistance to protein unfolding is observed for all iron(III) complexes compared to Cu(II) complexes of lactoferrin and transferrin as determined by isothermal unfolding and thermal denaturation. 5. Relative stabilization of the different metal-protein complexes investigated within this study were determined to be as follows: Lf-Fe(III) greater than Lf-Cu(II); Tf-Fe(III) greater than Tf-Cu(II), and Lf-Fe(III) greater than Tf-Fe(III); Lf-Cu(II) greater than Tf-Cu(II).     

Enhancement of cytotoxicity of bleomycin by dithiocarbamates: formation of bis(dithiocarbamato) cu(II).

Properties of the reactions of dithiocarbamates and their Cu(II) or Fe(III) complexes with Ehrlich cells were determined and related to their effects on the inhibition of cell proliferation caused by bleomycin and Cu bleomycin. In complete culture medium containing Eagle's minimal essential medium plus Earles salts and 2.5% fetal calf serum, dimethyl- and diethyldithiocarbamates and their copper complexes inhibit cell proliferation and cause cell death. The copper complexes are more effective agents. Ferric tris-diethyldithiocarbamate is also a cytotoxic species. In contrast, when cells are exposed to dimethyldithiocarbamate or its copper complex in Ringer's buffer under metal-restricted condition, washed, and then placed in complete medium, the copper complex is much more active in inhibiting cell growth. The difference is magnified when dihydroxyethyldithiocarbamate and N-methylglucamine dithiocarbamate and their copper complexes are compared in complete media. Incubation of bleomycin or copper bleomycin with Ehrlich cells in Ringer's buffer with or without dimethyldithiocarbamate or bis-dimethyldithiocarbamato Cu(II) leads to no enhancement of cytotoxicity from combinations of agents, except when the two copper complexes are present. Diethyl- or dimethyldithiocarbamate readily extracts copper from Cu(II)bleomycin and iron from Fe(III)bleomycin when ethylacetate is present to remove the tris-dithiocarbamato Fe(III) complex from aqueous solution. When bis-dimethyldithiocarbamato Cu(II) is incubated with Ehrlich cells, copper is released from the complex and bound to high molecular weight and metallothionein fractions. A reductive mode of dissociation of the copper complexes in cells is supported by ESR experiments. Reactions of diethyl- and dimethyldithiocarbamato Cu(II) with thiol compounds demonstrates one possible mechanism of reduction of these complexes.     

Metal complexes of the antibiotic nalidixic acid

The preparations and spectral properties are reported of a range of complexes of nalidixic acid (= HNal) with some metal ions in the series CrZn and also Mg, Ca, Cd, Hg and Pd(II). Most of the compounds formed by the divalent metal ions had a 2:1 HNal:metal ratio, and some of them are polymeric. Complexes in which the carboxylate group of HNal functions as a chelate were isolated with Cu(II), Pd(II), Fe(III) and Cr(III).