Detection of DNA Autoantibodies by Electrophoretic Mobility Shift Assay.

Although the presence of antibodies against double-stranded (ds) DNA is considered the serological hallmark of systemic lupus erythematosus (SLE), it is not detected in all SLE patients by routine laboratory tests. Looking at DNA-anti(ds)DNA interaction as one kind of DNA-protein interaction gave us the grounds for a novel type of assay, easy to perform, and providing a direct insight on DNA-anti(ds)DNA IgG interaction. The assay is an application of the electrophoretic mobility shift assay (EMSA) and is based on the observation that the electrophoretic mobility of a DNA-protein complex is typically less than that of free DNA. The EMSA, performed here with purified bacterial DNA and the purified IgG fraction of sera from systemic lupus erythematosus (SLE) or other patients as well as from healthy individuals, revealed itself to be more sensitive than the routinely used assays for the detection of anti-dsDNA in SLE and discoid lupus erythematosus (DLE) patients. In addition, besides providing a direct visualization of DNA-anti(ds)DNA IgG complexes, the assay offers the possibility to study in detail the nature of DNA-IgG interactions. In a further development, we showed that the assay could be performed successfully with sera.

DNA mobility shift assay as a tool for the detection of anti-dsDNA antibodies in sera from discoid lupus erythematosus patients.

Discoid lupus erythematosus (DLE) is a form of local inflammatory autoimmune disease limited to the skin, involving essentially the face, scalp and ear. DLE occurs in genetically predisposed individuals, sunlight being an identified trigger. Diagnosis is made by clinical examination and histopathology; laboratory tests occasionally performed include anti-nuclear antibodies titers and presence of circulating antibodies against dsDNA. DLE patients have about a 10% chance of developing systemic lupus erythematosus (SLE), a systemic inflammatory autoimmune disease affecting a range of internal organs. Although elevated titers of anti-dsDNA antibodies is an earmark for lupus disease, they are detected in only 20-55% of DLE patients by routine laboratory tests such as enzyme-linked immunosorbent assay (ELISA). In this research, we applied an electrophoretic mobility shift assay (EMSA) in parallel with an ELISA for the detection of circulating anti-dsDNA in DLE patients. The assays were conducted on sera as well as on the immunoglobulin G fraction from sera of 24 DLE patients and of 24 healthy individuals. The EMSA was positive for all DLE patients while the ELISA was positive for only 36% of them; both assays were negative for the healthy individuals. EMSA conducted on the sera of 15 patients with lichen planus was negative in all cases. Results suggest that the EMSA is more sensitive than the routine tests used for the detection of anti-dsDNA in DLE, thus helping to improve early detection of the disease and, by extension, to better evaluate the factors triggering the disease.

Acriflavine-mediated apoptosis and necrosis in yeast Candida utilis.

Acriflavine is an antiseptic, fungicide, and effective agent against parasitic infections, inducing petite mutation in the yeast Saccharomyces cerevisiae and kinetoplast loss in Trypanosomidae. Here we showed that acriflavine caused both apoptosis and necrosis in the yeast Candida utilis. Cells were cultured in minimal medium, with 1.5% ethanol as substrate, in the presence of 30-180 micromol/L acriflavine. Fluorescence measurements showed a linear concentration-dependence flux of the drug into the cells. Acriflavine induced a decrease in cell number, an increase in trypan blue-positive cells, and a decrease in cell viability. Cells cultured in the presence of acriflavine showed an alteration in their respiratory control ratio and a decrease in their cytochrome content. Fluorescence microscopy, after acridine orange staining, revealed the presence of apoptotic cells in cultures conducted in the presence of acriflavine. Electron microscopy of cells grown in the presence of acriflavine showed apoptotic cells exhibiting chromatin condensation, cytoplasmic lysis, but reasonably well-preserved mitochondria, whereas necrotic cells showed no distinctive intracellular organelles. Data showed that acriflavine caused both apoptosis and necrosis. Moreover, acriflavine induced oxidative phosphorylation uncoupling. Generally, apoptosis is considered to be mediated either by a change in mitochondrial permeability and cytochrome c release or by plasma membrane death receptor activation. The outer mitochondrial membrane permeability to cytochrome c, with efflux of protons to the cytosol and cytoplasmic acidification, produced a collapse in the electrochemical proton gradient, a decrease in ATP synthesis, and subsequent cytolysis leading to apoptosis and necrosis.

Structural and functional alterations of catalase induced by acriflavine, a compound causing apoptosis and necrosis.

Acriflavine is an antiseptic agent causing both apoptosis and necrosis in yeast. In this work, its effect on the structure and function of catalase, a vital enzyme actively involved in protection against oxidative stress, was investigated. In vitro kinetic studies showed that acriflavine inhibited the enzymatic activity in a competitive manner. The residual activity detectable after preincubation of catalase (1.5 nmol/L) with various concentrations of acriflavine went from 50% to 20% of the control value as the acriflavine concentration increased from 30 to 90 micromol/L. Correlatively with the decrease in activity, alterations in the enzyme's conformation were observed as indicated by fluorescence spectroscopy, circular dichroism spectroscopy, and electronic absorption spectroscopy. The enzyme's intrinsic fluorescence obtained upon excitation at either 297 nm (tryptophan residues) or 280 nm (tyrosine and tryptophan residues) decreased as a function of acriflavine concentration. Circular dichroism studies showed alterations of the protein structure by acriflavine with up to 13% decrease in alpha helix, 16% increase in beta-sheet content, 17% increase in random coil, and 4% increase in beta turns. Spectrophotometric studies showed a blueshift and modifications in the chromicity of catalase at 405 nm, corresponding to an absorbance band due to the enzyme's prosthetic group. Thus, acriflavine induced in vitro a profound change in the structure of catalase so that the enzyme could no longer function. Our results showed that acriflavine, a compound producing apoptosis and necrosis, can have a direct effect on vital functions in cells by disabling key enzymes.

Functional and structural alterations induced by copper in xanthine oxidase.

Xanthine oxidase (XO), a key enzyme in purine metabolism, produces reactive oxygen species causing vascular injuries and chronic heart failure. Here, copper's ability to alter XO activity and structure was investigated in vitro after pre-incubation of the enzyme with increasing Cu(2+) concentrations for various periods of time. The enzymatic activity was measured by following XO-catalyzed xanthine oxidation to uric acid under steady-state kinetics conditions. Structural alterations were assessed by electronic absorption, fluorescence, and circular dichroism spectroscopy. Results showed that Cu(2+) either stimulated or inhibited XO activity, depending on metal concentration and pre-incubation length, the latter also determining the inhibition type. Cu(2+)-XO complex formation was characterized by modifications in XO electronic absorption bands, intrinsic fluorescence, and alpha-helical and beta-sheet content. Apparent dissociation constant values implied high- and low-affinity Cu(2+) binding sites in the vicinity of the enzyme's reactive centers. Data indicated that Cu(2+) binding to high-affinity sites caused alterations around XO molybdenum and flavin adenine dinucleotide centers, changes in secondary structure, and moderate activity inhibition; binding to low affinity sites caused alterations around all XO reactive centers including FeS, changes in tertiary structure as reflected by alterations in spectral properties, and drastic activity inhibition. Stimulation was attributed to transient stabilization of XO optimal conformation. Results also emphasized the potential role of copper in the regulation of XO activity stemming from its binding properties.

Effect of ionic detergents, nonionic detergents, and chaotropic agents on polyphenol oxidase activity from dormant saffron (Crocus sativus L.) corms.

Polyphenol oxidase (PPO; EC 1.14.18.1) catalyzes the hydroxylation of monophenols to o-diphenols (cresolase activity) and the oxidation of o-diphenols to o-quinones (catecholase activity), leading to browning in plants and produce. Further interest in the enzyme has been triggered by the active role that it plays in plant defense systems. PPO can be found in latent forms and is activated in vitro by various agents including urea, detergents, and proteases. The activation of PPO from several sources by sodium dodecyl sulfate (SDS) has been extensively investigated, but reports on the effect of other detergents or on the differential effect of detergents on each of PPO's activities are scarce. In addition, investigations on the enzyme in other plant parts besides fruits and vegetables are also scarce. Here, the effect of various detergents and chaotropic agents on PPO from dormant saffron (Crocus sativus L.) corm extract was investigated. SDS and sarkosyl activated the cresolase activity, while only SDS activated the catecholase activity. All other detergents tested, in milli- or micromolar concentrations, inhibited the cresolase activity but barely affected the catecholase activity. In contrast, urea and guanidine-HCl drastically inhibited the catecholase activity but moderately inhibited the cresolase activity. The same effects were obtained on the partially purified enzyme. Results identified a PPO, present in dormant corms, which was activated only by anionic detergents and was inhibited by other reputed activating agents such as urea. Results also emphasized the differences in structure and accessibility of the active sites for cresolase and catecholase activities.

Sensitivity to detergents and plasmid curing in Enterococcus faecalis.

This research reports the sensitivity of a clinical isolate of Enterococcus faecalis to sodium N-lauroylsarcosinate (sarkosyl) and sodium dodecyl sulfate (SDS), as well as the efficiency of these detergents in curing the strain. Compared to Escherichia coli, Enterococcus faecalis was very sensitive to both detergents, with minimum inhibitory concentrations (MIC) for the latter being 100 times lower than for Escherichia coli. The clinical isolate of Enterococcus faecalis used in this study exhibited plasmid-borne resistance to kanamycin (MIC 2 mg/ml) and tetracycline (MIC 50 mug/ml); 3% curing was observed after growth in the presence of sarkosyl but no curing was observed after growth in the presence of either SDS or acridine orange. In contrast, 35% curing of plasmid-bearing Escherichia coli was observed after growth in the presence of either SDS or acridine orange, but none was observed after growth in the presence of sarkosyl.

DNA strand breaks by metal-induced oxygen radicals in purified Salmonella typhimurium DNA.

Purified Salmonella typhimurium DNA was incubated for 1h at 37 degrees C with various concentrations (10-100 muM) of transition metal ions (Fe(2+), Fe(3+), Cu(2+), Ni(2+), Cd(2+)), with various concentrations (0.1-100 mM) of H(2)O(2), and with various concentrations of each transition metal ion in the presence of various concentrations of H(2)O(2). Damage to DNA was assessed by electrophoresis of the reaction mixtures in 1% agarose gel. Breakage of the DNA strands would produce a series of DNA fragments resulting in a smear in the gel, while intact DNA produced a single band. Results showed that no damage to the DNA was detectable after incubation with either H(2)O(2) alone or either of the metal ions alone. However, all of the metal ions investigated triggered DNA breakage in the presence of H(2)O(2). The extent of breakage depended on the metal ion and on its concentration, as well as on the H(2)O(2) concentration. Addition of either EDTA or catalase to the reaction mixture completely inhibited the DNA degradation, confirming the involvement of both the metal ion and the H(2)O(2) in the breakage of DNA strands. Production of the hydroxyl radical when H(2)O(2) and a metal ion were both present in the reaction mixture was evidenced by the thiobarbituric acid method. The most extensive damage was caused by Cu(2+) followed, in decreasing order, by Fe(2+), Fe(3+), Ni(2+), and Cd(2+).

Antioxidant enzymes during hypoxia-anoxia signaling events in Crocus sativus L. corm.

The activity of reactive oxygen species (ROS)-scavenging enzymes, catalase, superoxide dismutase (SOD), glutathione peroxidase, o-dianisidine and ascorbate peroxidases, was investigated in Crocus sativus L. corms cultivated in normoxic and hypoxic-anoxic conditions. The activity of the ROS-scavenging enzymes studied varied during cultivation. However, the pattern of ROS-scavenging enzymes production was different in corms cultivated in normoxic and hypoxic-anoxic conditions. In normoxic conditions, only the activities of peroxidases and SOD were stimulated. In dormant corms placed under hypoxia-anoxia, the activities of catalase, SOD, and glutathione peroxidase were stimulated, with the highest stimulation observed for catalase, followed by SOD, and then glutathione peroxidase. In corms that had been rooted for 3 days before being placed in hypoxia-anoxia, the activities of all ROS-scavenging enzymes studied were stimulated with the highest stimulation still observed for catalase, followed by the peroxidases, and finally SOD. Thus catalase was the prevailing enzyme produced under hypoxia-anoxia.

Stepwise binding of nickel to horseradish peroxidase and inhibition of the enzymatic activity.

The incubation of horseradish peroxidase C (HRPC) with millimolar concentrations of nickel, at room temperature and at pH 4.0, induced the progressive formation of a metal-enzyme complex characterized by alterations of the enzyme Soret absorption band that were time- as well as nickel concentration- dependent. For any given incubation period between 1 and 60 min, 2 values for the apparent dissociation constant (K(d)) were found, suggesting the presence of binding sites with different affinities for nickel. The value of each K(d) dropped as the incubation time increased, indicating a progressive stabilization of the metal-enzyme complex. Hill plots suggested a cooperative binding of up to four Ni2+ ions per molecule of HRPC. The inhibition of the enzymatic activity by nickel was studied by following the H2O2-mediated oxidation of o-dianisidine by HRPC under steady-state kinetic conditions. Ni2+ was found to be either a noncompetitive or a mixed inhibitor of HRPC depending both on the duration of preincubation with the enzyme and on Ni2+ concentration. The enzyme remained active only over a limited metal concentration range and data indicated that binding of one Ni2+ affected the substrate binding site, binding of a second Ni2+ affected both substrate and peroxide binding sites, and binding of more than 2 Ni2+ per HRPC molecule led to complete loss of enzymatic activity. Results pointed to the damaging effects of prolonged exposure to heavy metals and also to the existence of a critical metal concentration beyond which immediate abolishing of enzymatic activity was observed.

Plasma membrane alteration is an early signaling event in doxorubicin-induced apoptosis in the yeast Candida utilis.

Signaling pathways such as increased ceramide, mitochondrial dysfunction, and P3 and caspase activation are produced by anticancer drugs and lead to apoptosis. In this research we show that the first event after culturing the yeast Candida utilis in the presence of low doses of doxorubicin (25 microg/mL) is the morphological alteration of the plasma membrane. In the presence of higher doxorubicin doses (>/=50 microg/mL), in addition to profound alterations in the plasma membrane, changes in mitochondrial shape and cristae organization were observed. Concomitantly, increases in respiration, substrate oxidation, and cytochrome biosynthesis were observed at low doxorubicin doses (up to 25 microg/mL), whereas a progressive decrease was observed at higher doses. [(3)H]Leu incorporation into proteins increased by 40% in the mitochondrial fraction and by 19% in the cytosol in the presence of 25 microg/mL doxorubicin; it decreased to 80% of the control in the cytosol in the presence of 1 mg/mL doxorubicin. Morphologically, doxorubicin doses of up to 200 microg/mL produced apoptosis, whereas higher doxorubicin doses produced necrosis.

Hypoxia/anoxia as signaling for increased alcohol dehydrogenase activity in saffron (Crocus sativus L.) corm.

Alcohol dehydrogenase, NAD-dependent lactate dehydrogenase, and NAD-independent lactate dehydrogenase activities were investigated in corms cultivated in normoxic and hypoxic/anoxic conditions. Depending on the developmental stage, hypoxia/anoxia was a signal for increase in either alcohol dehydrogenase or NAD-dependent lactate dehydrogenase. NAD-independent lactate dehydrogenase contributed to the recycling of lactate, thus preventing acidosis.

Heterogeneous inhibition of horseradish peroxidase activity by cadmium.

Inhibition of horseradish peroxidase (HRP) activity by cadmium was studied under steady-state kinetic conditions after preincubation of the enzyme with millimolar concentrations of Cd(2+) for various periods of time. The H(2)O(2)-mediated oxidation of o-dianisidine by HRP was used to assess the enzymatic activity. Cd(2+) was found to be either a noncompetitive inhibitor of HRP or a mixed inhibitor of HRP depending both on the duration of incubation with HRP and on Cd(2+) concentration. Furthermore, for the same inhibition type, K(i) values dropped as incubation time increased. These results suggested that Cd(2+) would slowly bind to the enzyme and progressively induce conformational changes. Spectrophotometric analysis showed that indeed Cd(2+) altered the heme Soret absorption band on binding HRP and exhibited a K(d) which decreased as the incubation time of HRP with Cd(2+) increased. Hill plots suggested a cooperative binding of up to three Cd(2+) ions per molecule of HRP. Thus, Cd(2+) binding to HRP resulted in progressive inhibition of enzymatic activity with a change in the inhibition type as the number of Cd(2+) ions per HRP molecule increased. Results also illustrated the potential danger of long-term exposure to heavy metals, even for enzymes with low affinity for them.