Human colon adenocarcinoma HT-29 cell: electrochemistry and nicotine stimulation.

Recently, it was demonstrated that colorectal cancer HT-29 cells can secrete epinephrine (adrenaline) in an autocrine manner to auto-stimulate cellular growth by adrenoreceptors activation, and that this secretion is enhanced by nicotine, showing an indirect relation between colorectal cancer and tobacco. The electrochemical behaviour of human colon adenocarcinoma HT-29 cells from a colorectal adenocarcinoma cell line, the hormone and neurotransmitter epinephrine, and nicotine, were investigated by cyclic voltammetry, using indium tin oxide (ITO), glassy carbon (GC) and screen printed carbon (SPC) electrodes. The oxidation of the HT-29 cells, previously grown onto ITO or SPC surfaces, followed an irreversible oxidation process that involved the formation of a main oxidation product that undergoes irreversible reduction, as in the epinephrine oxidation mechanism. The effect of nicotine stimulation of the HT-29 cells was also investigated. Nicotine, at different concentration levels 1, 2 and 15 mM, was introduced in the culture medium and an increase with incubation time, 0 to 3h and 30 min, of the HT-29 cells oxidation and reduction peaks was observed. The interaction of nicotine with the HT-29 cells stimulated the epinephrine secretion causing an increase in epinephrine release concentration, and enabling the conclusion that epinephrine and nicotine play an important role in the colorectal tumour growth.

Effects of low molecular weight sulfated galactan fragments from Botryocladia occidentalis on the pharmacological and enzymatic activity of sPLA2 from Crotalus durissus cascavella.

Low molecular weight fragments of sulfated galactans (Boc-5 and Boc-10) from the red algae Botryocladia occidentalis significantly inhibited Crotalus durissus cascavella sPLA2 enzymatic activity. Equimolar ratios of sPLA2 to Boc-5 or Boc-10 resulted in allosteric inhibition of sPLA2. Under the conditions tested, we observed that both Boc-5 and Boc-10 strongly decreased edema, myonecrosis, and neurotoxicity induced by native sPLA2.

Effect of umbelliferone (7-hydroxycoumarin, 7-HOC) on the enzymatic, edematogenic and necrotic activities of secretory phospholipase A2 (sPLA2) isolated from Crotalus durissus collilineatus venom.

Flavonoids, coumarins and other polyphenolic compounds are powerful antioxidants both in hydrophilic and lipophylic environments with diverse pharmacological properties including anti-inflammatory activity. Despite being widely used as powerful therapeutic agents for blood coagulation disorders, more specifically to control some serine protease enzymes, the mechanism of anti-inflammatory activity of coumarins is unknown, unlike that of flavonoids. Although their controlling effect on serine proteases is well acknowledged, their action on secretory phospholipase A2 (sPLA2) remains obscure. The present study describes the interaction between umbelliferone (7-HOC) and the sPLA2 from Crotalus durissus collilineatus venom. In vitro inhibition of sPLA2 enzymatic activity by 7-HOC was estimated using 4N3OBA as substrate, resulting in an irreversible decrease in such activity proportional to 7-HOC concentration. The biophysical interaction between 7-HOC and sPLA2 was examined by fluorescent spectral analysis and circular dichroism studies. Results from both techniques clearly showed that 7-HOC strongly modified the secondary structure of this enzyme and CD spectra revealed that it strongly decreased sPLA2 alpha-helical conformation. In addition, two-dimensional electrophoresis indicated an evident difference between HPLC-purified native and 7-HOC-treated sPLA2s, which were used in pharmacological experiments to compare their biological activities. In vivo anti-inflammatory activity was assessed by the sPLA2-induced mouse paw edema model, in which 7-HOC presented an effect similar to those of dexamethasone and cyproheptadine against the pro-inflammatory effect induced by native sPLA2 on the mouse paw edema, mast cell degranulation and skin edema. On the other hand, 7-HOC exhibited a more potent inhibitory effect on sPLA2 than that of p-bromophenacyl bromide (p-BPB). Our data suggest that 7-HOC interacts with sPLA2 and causes some structural modifications that lead to a sharp decrease or inhibition of the edematogenic and myotoxic activities of this enzyme, indicating its potential use to suppress inflammation induced by sPLA2 from the snake venom.

In situ electrochemical and AFM study of thalidomide-DNA interaction.

The interaction of thalidomide (TD) with double-stranded DNA (dsDNA) was studied using atomic force microscopy (AFM) at highly oriented pyrolytic graphite (HOPG), differential pulse voltammetry (DPV) at glassy carbon electrodes (GCE), UV-Vis and electrophoresis. After incubation of dsDNA with different concentrations of TD, the AFM images show the formation of thin and incomplete TD-DNA network films with a number of embedded molecular aggregates and regions of uncovered HOPG. Both the TD-dsDNA aggregates and network thickness directly depended on the TD concentration and incubation time. The voltammetric data also showed that the modifications caused by TD to the DNA double helical structure are time-dependent. In agreement with AFM, DPV, UV-Vis and electrophoresis results, a model is proposed for the TD-DNA interaction, considering that TD intercalates into the dsDNA, causing defects in the dsDNA secondary structure and DNA double helix unwinding. Moreover, both AFM and DPV show that condensation is caused to DNA by TD and occurs until 24 h of incubation, as well as DNA oxidative damage, detected electrochemically by the appearance of the 8-oxoGua and/or 2,8 oxoAde oxidation peak.

Enzymatic and structural characterization of new PLA2 isoform isolated from white venom of Crotalus durissus ruruima.

This work reports the structural and enzymatic characterization of a new sPLA2 from the white venom of Crotalus durissus ruruima, nominated PLA2A. The homogeneity of the PLA2A fraction and its molecular mass were initially evaluated by SDS-PAGE and confirmed by MALDI-TOF spectrometry, indicating a molecular mass of 14,299.34Da. Structural investigation, through circular dichroism spectroscopy, revealed that PLA2A has a high content of alpha helix and beta-turn structures, 45.7% and 35.6% respectively. Its amino acid sequence, determined by Edman degradation and "de novo amino acid sequencing", exhibited high identity to PLA2 Cdt F15 from Crotalus durissus terrificus. The enzymatic investigation, conducted using the synthetic substrate 4-nitro-3-(octanoyloxy)-benzoic acid, determined its V(max) (7.56nmoles/min) and K(M) (2.76mM). Moreover, PLA2A showed an allosteric behavior and its enzymatic activity was dependent on Ca(2+). Intrinsic fluorescence measurements suggested that Ca(2+) induced a significant increase of PLA2A fluorescence, whereas its replacement for Mg(2+), Mn(2+), Sn(2+) and Cd(2+) apparently induced no structural modifications. The optimal pH and temperature for the enzymatic activity of PLA2A were 8.4 and 40 degrees C, respectively, and the minimal concentration of p-BPB and crotapotin that significantly inhibited such activity was 0.75mM and 0.4muM, respectively. In addition, PLA2A showed a significant antibacterial effect that was not strictly dependent on the enzymatic activity of such sPLA2.

In situ evaluation of heavy metal-DNA interactions using an electrochemical DNA biosensor.

Heavy metal ions, lead, cadmium and nickel, are well known carcinogens with natural different origins and their direct mode of action is still not fully understood. A dsDNA-electrochemical biosensor, employing differential pulse voltammetry, was used for the in situ evaluation of Pb2+, Cd2+ and Ni2+ interaction with dsDNA. The results confirm that Pb2+, Cd2+ and Ni2+ bind to dsDNA, and that this interaction leads to different modifications in the dsDNA structure. These modifications were electrochemically recognized as changes in the oxidation peaks of guanosine and adenosine bases. Using homopolynucleotides of guanine and adenine it has been proved that the interaction between Pb2+ and DNA causes oxidative damage and preferentially takes place at adenine-containing segments, with the formation of 2,8-dihydroxyadenine, the oxidation product of adenine residues and a biomarker of DNA oxidative damage. The Pb2+ bound to dsDNA can still undergo oxidation. The interaction of Cd2+ and Ni2+ causes conformational changes, destabilizing the double helix, which can enable the action of other oxidative agents on DNA.

Electrochemical oxidation of ochratoxin A at a glassy carbon electrode and in situ evaluation of the interaction with deoxyribonucleic acid using an electrochemical deoxyribonucleic acid-biosensor.

Ochratoxin A (OTA) is a fungal metabolite that occurs in foods, beverages, animal tissues, human blood and presents carcinogenic, teratogenic and nephrotoxic properties. This study concerns the redox properties of OTA using electrochemical techniques which have the potential for providing insights into the biological redox reactions of this molecule. The in situ evaluation of the OTA interaction with DNA using a DNA-electrochemical biosensor is also reported. The oxidation of OTA is an irreversible process proceeds with the transfer of one electron and one proton in a diffusion-controlled mechanism. The diffusion coefficient of OTA was calculated in pH 7 phosphate buffer to be D(O) = 3.65x10(-6) cm2 s(-1). The oxidation of OTA is also pH dependent for electrolytes with pH<7 and involves the formation of a main oxidation product which adsorbs strongly at the GCE surface undergoing reversible oxidation. In alkaline electrolytes OTA undergoes chemical deprotonation, the oxidation involving only the transfer of one electron. The electrochemical dsDNA-biosensor was also used to evaluate the possible interaction between OTA and DNA. The experiments have clearly proven that OTA interacts and binds to dsDNA strands immobilized onto a GCE surface, but no evidence of DNA-damage caused by OTA was obtained.