Ana V. Coelho

Phenolic Compounds and Antioxidant Activity of Olea europaea L. Fruits and Leaves

Extracts of leaves, fruits and seeds of olive tree cultivars of Trás-os-Montes e Alto Douro (Portugal) were analysed by reverse phase HPLC with diode array detection and mass spectrometry (MS). This methodology allowed the identification of some common phenolic compounds, namely, verbascoside, rutin, luteolin-7-glucoside, oleuropein and hydroxytyrosol. Moreover the hyphenation of HPLC with MS enabled the identification of nüzhenide in olive seeds. An oleuropein glucoside was also detected in olive tree leaves. The total phenolic content was determined with the Folin Denis reagent and the total antioxidant activity with the ABTS method. There is a correlation between total antioxidant activity and total phenolic content with the exception of the seed extracts analysed. The apparent high antioxidant activity of seed extracts may be due to nüzhenide, a secoiridoid that is the major phenolic component of olive seeds. These results suggest a possible application of olive seeds as sources of natural antioxidants.

Enzymatic biotransformation of the azo dye Sudan Orange G with bacterial CotA-laccase

In the present study we show that recombinant bacterial CotA-laccase from Bacillus subtilis is able to decolourise, at alkaline pH and in the absence of redox mediators, a variety of structurally different synthetic dyes. The enzymatic biotransformation of the azo dye Sudan Orange G (SOG) was addressed in more detail following a multidisciplinary approach. Biotransformation proceeds in a broad span of temperatures (30-80 degrees C) and more than 98% of Sudan Orange G is decolourised within 7h by using 1 U mL(-1) of CotA-laccase at 37 degrees C. The bell-shape pH profile of the enzyme with an optimum at 8, is in agreement with the pH dependence of the dye oxidation imposed by its acid-basic behavior as measured by potentiometric and electrochemical experiments. Seven biotransformation products were identified using high-performance liquid chromatography and mass spectrometry and a mechanistic pathway for the azo dye conversion by CotA-laccase is proposed. The enzymatic oxidation of the Sudan Orange G results in the production of oligomers and, possibly polymers, through radical coupling reactions. A bioassay based on inhibitory effects over the growth of Saccharomyces cerevisiae shows that the enzymatic bioremediation process reduces 3-fold the toxicity of Sudan Orange G.

Desulfoferrodoxin structure determined by MAD phasing and refinement to 1.9-Å resolution reveals a unique combination of a tetrahedral FeS4 centre with a square pyramidal FeSN4 centre

Abstract The structure of desulfoferrodoxin (DFX), a protein containing two mononuclear non-heme iron centres, has been solved by the MAD method using phases determined at 2.8 Å resolution. The iron atoms in the native protein were used as the anomalous scatterers. The model was built from an electron density map obtained after density modification and refined against data collected at 1.9 Å. Desulfoferrodoxin is a homodimer which can be described in terms of two domains, each with two crystallographically equivalent non-heme mononuclear iron centres. Domain I is similar to desulforedoxin with distorted rubredoxin-type centres, and domain II has iron centres with square pyramidal coordination to four nitrogens from histidines as the equatorial ligands and one sulfur from a cysteine as the axial ligand. Domain I in DFX shows a remarkable structural fit with the DX homodimer. Furthermore, three β-sheets extending from one monomer to another in DFX, two in domain I and one in domain II, strongly support the assumption of DFX as a functional dimer. A calcium ion, indispensable in the crystallisation process, was assumed at the dimer interface and appears to contribute to dimer stabilisation. The C-terminal domain in the monomer has a topology fold similar to that of fibronectin III.

Iminoboronates: a new strategy for reversible protein modification.

Protein modification has entered the limelight of chemical and biological sciences, since, by appending small molecules into proteins surfaces, fundamental biological and biophysical processes may be studied and even modulated in a physiological context. Herein we present a new strategy to modify the lysines ε-amino group and the proteins N-terminal, based on the formation of stable iminoboronates in aqueous media. This functionality enables the stable and complete modification of these amine groups, which can be reversible upon the addition of fructose, dopamine, or glutathione. A detailed DFT study is also presented to rationalize the observed stability toward hydrolysis of the iminoboronate constructs.

Quantification of Free Circulating Tumor DNA as a Diagnostic Marker for Breast Cancer

AIM To determine whether the amounts of circulating DNA could discriminate between breast cancer patients and healthy individuals by using real-time PCR quantification methodology. METHODS Our standard protocol for quantification of cell-free plasma DNA involved 175 consecutive patients with breast cancer and 80 healthy controls. RESULTS We found increased levels of circulating DNA in breast cancer patients compared to control individuals (105.2 vs. 77.06 ng/mL, p < 0.001). We also found statistically significant differences in circulating DNA amounts in patients before and after breast surgery (105.2 vs. 59.0 ng/mL, p = 0.001). Increased plasma cell-free DNA concentration was a strong risk factor for breast cancer, conferring an increased risk for the presence of this disease (OR, 12.32; 95% CI, 2.09-52.28; p < 0.001). CONCLUSIONS Quantification of circulating DNA by real-time PCR may be a good and simple tool for detection of breast cancer with a potential to clinical applicability together with other current methods used for monitoring the disease.

Hydrogenases in Desulfovibrio vulgaris Hildenborough: structural and physiologic characterisation of the membrane-bound [NiFeSe] hydrogenase

The genome of Desulfovibrio vulgaris Hildenborough (DvH) encodes for six hydrogenases (Hases), making it an interesting organism to study the role of these proteins in sulphate respiration. In this work we address the role of the [NiFeSe] Hase, found to be the major Hase associated with the cytoplasmic membrane. The purified enzyme displays interesting catalytic properties, such as a very high H2 production activity, which is dependent on the presence of phospholipids or detergent, and resistance to oxygen inactivation since it is isolated aerobically in a Ni(II) oxidation state. Evidence was obtained that the [NiFeSe] Hase is post-translationally modified to include a hydrophobic group bound to the N-terminal, which is responsible for its membrane association. Cleavage of this group originates a soluble, less active form of the enzyme. Sequence analysis shows that [NiFeSe] Hases from Desulfovibrionacae form a separate family from the [NiFe] enzymes of these organisms, and are more closely related to [NiFe] Hases from more distant bacterial species that have a medial [4Fe4S]2+/1+ cluster, but not a selenocysteine. The interaction of the [NiFeSe] Hase with periplasmic cytochromes was investigated and is similar to the [NiFe]1 Hase, with the Type I cytochrome c3 as the preferred electron acceptor. A model of the DvH [NiFeSe] Hase was generated based on the structure of the Desulfomicrobium baculatum enzyme. The structures of the two [NiFeSe] Hases are compared with the structures of [NiFe] Hases, to evaluate the consensual structural differences between the two families. Several conserved residues close to the redox centres were identified, which may be relevant to the higher activity displayed by [NiFeSe] Hases.

Insulin glycation by methylglyoxal results in native-like aggregation and inhibition of fibril formation

BackgroundInsulin is a hormone that regulates blood glucose homeostasis and is a central protein in a medical condition termed insulin injection amyloidosis. It is intimately associated with glycaemia and is vulnerable to glycation by glucose and other highly reactive carbonyls like methylglyoxal, especially in diabetic conditions. Protein glycation is involved in structure and stability changes that impair protein functionality, and is associated with several human diseases, such as diabetes and neurodegenerative diseases like Alzheimers disease, Parkinsons disease and Familiar Amyloidotic Polyneuropathy. In the present work, methylglyoxal was investigated for their effects on the structure, stability and fibril formation of insulin.ResultsMethylglyoxal was found to induce the formation of insulin native-like aggregates and reduce protein fibrillation by blocking the formation of the seeding nuclei. Equilibrium-unfolding experiments using chaotropic agents showed that glycated insulin has a small conformational stability and a weaker dependence on denaturant concentration (smaller m-value). Our observations suggest that methylglyoxal modification of insulin leads to a less compact and less stable structure that may be associated to an increased protein dynamics.ConclusionsWe propose that higher dynamics in glycated insulin could prevent the formation of the rigid cross-β core structure found in amyloid fibrils, thereby contributing to the reduction in the ability to form fibrils and to the population of different aggregation pathways like the formation of native-like aggregates.

Yeast protein glycation in vivo by methylglyoxal Molecular modification of glycolytic enzymes and heat shock proteins

Protein glycation by methylglyoxal is a nonenzymatic post‐translational modification whereby arginine and lysine side chains form a chemically heterogeneous group of advanced glycation end‐products. Methylglyoxal‐derived advanced glycation end‐products are involved in pathologies such as diabetes and neurodegenerative diseases of the amyloid type. As methylglyoxal is produced nonenzymatically from dihydroxyacetone phosphate and d‐glyceraldehyde 3‐phosphate during glycolysis, its formation occurs in all living cells. Understanding methylglyoxal glycation in model systems will provide important clues regarding glycation prevention in higher organisms in the context of widespread human diseases. Using Saccharomyces cerevisiae cells with different glycation phenotypes and MALDI‐TOF peptide mass fingerprints, we identified enolase 2 as the primary methylglyoxal glycation target in yeast. Two other glycolytic enzymes are also glycated, aldolase and phosphoglycerate mutase. Despite enolases activity loss, in a glycation‐dependent way, glycolytic flux and glycerol production remained unchanged. None of these enzymes has any effect on glycolytic flux, as evaluated by sensitivity analysis, showing that yeast glycolysis is a very robust metabolic pathway. Three heat shock proteins are also glycated, Hsp71/72 and Hsp26. For all glycated proteins, the nature and molecular location of some advanced glycation end‐products were determined by MALDI‐TOF. Yeast cells experienced selective pressure towards efficient use of d‐glucose, with high methylglyoxal formation as a side effect. Glycation is a fact of life for these cells, and some glycolytic enzymes could be deployed to contain methylglyoxal that evades its enzymatic catabolism. Heat shock proteins may be involved in proteolytic processing (Hsp71/72) or protein salvaging (Hsp26).

The primary and three-dimensional structures of a nine-haem cytochrome c from Desulfovibrio desulfuricans ATCC 27774 reveal a new member of the Hmc family

BACKGROUND Haem-containing proteins are directly involved in electron transfer as well as in enzymatic functions. The nine-haem cytochrome c (9Hcc), previously described as having 12 haem groups, was isolated from cells of Desulfovibrio desulfuricans ATCC 27774, grown under both nitrate- and sulphate-respiring conditions. RESULTS Models for the primary and three-dimensional structures of this cytochrome, containing 292 amino acid residues and nine haem groups, were derived using the multiple wavelength anomalous dispersion phasing method and refined using 1.8 A diffraction data to an R value of 17.0%. The nine haem groups are arranged into two tetrahaem clusters, with Fe-Fe distances and local protein fold similar to tetrahaem cytochromes c3, while the extra haem is located asymmetrically between the two clusters. CONCLUSIONS This is the first known three-dimensional structure in which multiple copies of a tetrahaem cytochrome c3-like fold are present in the same polypeptide chain. Sequence homology was found between this cytochrome and the C-terminal region (residues 229-514) of the high molecular weight cytochrome c from Desulfovibrio vulgaris Hildenborough (DvH Hmc). A new haem arrangement in domains III and IV of DvH Hmc is proposed. Kinetic experiments showed that 9Hcc can be reduced by the [NiFe] hydrogenase from D. desulfuricans ATCC 27774, but that this reduction is faster in the presence of tetrahaem cytochrome c3. As Hmc has never been found in D. desulfuricans ATCC 27774, we propose that 9Hcc replaces it in this organism and is therefore probably involved in electron transfer across the membrane.

Mechanisms of Prostate Atrophy after Glandular Botulinum Neurotoxin Type A Injection: An Experimental Study in the Rat

BACKGROUND Previous studies in humans, dogs, and rats have shown that intraprostatic injection of botulinum neurotoxin type A (BoNTA) reduces gland size. OBJECTIVE To investigate the role of eventual impairment of sympathetic, parasympathetic, and sensory nerves to gland atrophy after intraprostatic BoNTA administration. DESIGN, SETTING, AND PARTICIPANTS Adult male Wistar rats weighing 300-350 g were used. INTERVENTION Animals were injected in the prostate ventral lobes with 0.2 ml of saline (n=6) or the same volume containing 10 U BoNTA (BOTOX) (n=18). Six rats treated with BoNTA further received the adrenergic agent phenylephrine (PHE, 0.05 mg/kg per day), six received the cholinergic drug bethanechol (2 mg/kg per day), and six received subcutaneous saline. Animals were sacrificed 1 wk later. MEASUREMENTS Prostates were weighed, fixed, and stained for sympathetic (tyrosine hydroxylase [TH]), parasympathetic (vesicular acetylcholine [ACh] transporter [VAChT]), and sensory nerve (calcitonin gene-related peptide [CGRP]) visualisation. Terminal deoxynucleotidyl transferase biotin-dUTP nick-end labelling (TUNEL) reaction was performed to investigate apoptosis. RESULTS AND LIMITATIONS Prostate weight in controls was 1.82+/-0.24 mg/100 g of rat weight. In BoNTA-treated rats, weight decreased to 1.28+/-0.18 mg /100 g of rat weight (p=0.002). In BoNTA plus PHE-treated rats, prostate weight was similar to controls: 1.78+/-0.27 (p=0.87). In rats treated with BoNTA plus bethanechol, weight was less than controls: 1.41+/-0.17 (p=0.01). The number of TH-positive fibres was markedly reduced after BoNTA (p<0.001). VAChT- and CGRP-positive fibres were scarce in controls, preventing further evaluation. Rats treated with BoNTA had more TUNEL-positive cells than controls (p<0.001) and rats treated with BoNTA plus PHE (p<0.001). There were no differences between the BoNTA and BoNTA plus bethanechol groups (p=0.81). Although showing atrophy after BoNTA injection, rat prostates do not develop benign prostatic hyperplasia (BPH). Thus, present findings should be used cautiously to explain prostate atrophy seen in men with BPH treated with BoNTA. CONCLUSIONS Prostate atrophy induced by BoNTA in the rat may be the result of sympathetic nerve impairment and decreased adrenergic stimulation of the gland. Data indirectly suggest that sympathetic drive plays a role in prostate-size regulation.