M.R.M. Domingues

Phenolic characterization of Northeast Portuguese propolis: usual and unusual compounds.

In this study, an ethanolic extract from Portuguese propolis was prepared, fractionated by high-performance liquid chromatography, and the identification of the phenolic compounds was done by electrospray mass spectrometry in the negative mode. This technical approach allowed the identification of 37 phenolic compounds, which included not only the typical phenolic acids and flavonoids found in propolis from temperate zones but also several compounds in which its occurrence have never been referred to in the literature. Four of the novel phenolic compounds were methylated and/or esterified or hydroxylated derivatives of common poplar flavonoids, although six peculiar derivatives of pinocembrin/pinobanksin, containing a phenylpropanoic acid derivative moiety in their structure, were also identified. Furthermore, the Portuguese propolis sample was shown to contain a p-coumaric ester derivative dimer.

Binding of epigallocatechin‐3‐gallate to transthyretin modulates its amyloidogenicity

MINT‐7294529: TTR (uniprotkb:P02766) and TTR (uniprotkb:P02766) bind (MI:0407) by comigration in non‐denaturing gel electrophoresis (MI:0404)

Phenolic Profiling of Portuguese Propolis by LC-MS Spectrometry: Uncommon Propolis Rich in Flavonoid Glycosides

INTRODUCTION Propolis is a chemically complex resinous substance collected by honeybees (Apis mellifera) from tree buds, comprising plant exudates, secreted substances from bee metabolism, pollen and waxes. Its chemical composition depends strongly on the plant sources available around the beehive, which have a direct impact in the quality and bioactivity of the propolis. Being as Portugal is a country of botanical diversity, the phenolic characterisation of propolis from the different regions is a priority. OBJECTIVE Extensive characterisation of the phenolic composition of Portuguese propolis from different continental regions and islands. METHOD Forty propolis ethanolic extracts were analysed extensively by liquid chromatography with diode-array detection coupled to electrospray ionisation tandem mass spectrometry (LC-DAD-ESI-MS(n) ). RESULTS Seventy-six polyphenols were detected in the samples and two groups of propolis were established: the common temperate propolis, which contained the typical poplar phenolic compounds such as flavonoids and their methylated/esterified forms, phenylpropanoid acids and their esters, and an uncommon propolis type with an unusual composition in quercetin and kaempferol glycosides - some of them never described in propolis. CONCLUSION The method allowed the establishment of the phenolic profile of Portuguese propolis from different geographical locations, and the possibility to use some phenolic compounds, such as kaempferol-dimethylether, as geographical markers. Data suggest that other botanical species in addition to poplar trees can be important sources of resins for Portuguese propolis.

Amphiphilic phthalocyanine-cyclodextrin conjugates for cancer photodynamic therapy.

Three phthalocyanines (Pcs) conjugated with α-, β- and γ-cyclodextrins (CDs) were prepared and their application as photosensitizer (PS) agents was assessed by photophysical, photochemical and in vitro photobiological studies. The photoactivity of Pc-α-CD and Pc-γ-CD ensures their potential as PDT drugs against UM-UC-3 human bladder cancer cells.

The enhancement of the cellulolytic activity of cellobiohydrolase I and endoglucanase by the addition of cellulose binding domains derived from Trichoderma reesei

The effect of isolated cellulose binding domains (CBDs) on the hydrolysis of filter paper and microcrystalline cellulose by both cellobiohydrolase I and endoglucanase, was studied. CBDs were obtained by proteolysis from cellulases using a scaled-up variant of our previous method. Experiments were performed for different enzyme/substrate ratios in both the absence and presence of CBDs. Hydrolysis of filter paper by intact cellobiohydrolase I in the presence of additional CBDs was found to have a synergistic effect, leading to an increase of the sugar production of up to 30%. The effect was less pronounced using microcrystalline cellulose, where an increase up to 16% was observed. Similar trends were found during the hydrolysis of both substrates by endoglucanase.

Following Healthy Pregnancy by NMR Metabolomics of Plasma and Correlation to Urine

This work presents the first NMR metabolomics study of maternal plasma during pregnancy, including correlation between plasma and urine metabolites. The expected decrease in circulating amino acids early in pregnancy was confirmed with six amino acids being identified as required by the fetus in larger extents. Newly observed changes in citrate, lactate, and dimethyl sulfone suggested early adjustments in energy and gut microflora metabolisms. Alterations in creatine levels were also noted, in addition to creatinine variations reflecting alterations in glomerular filtration rate. Regarding plasma macromolecules, HDL and LDL+VLDL levels were confirmed to increase throughout pregnancy, although at different rates and accompanied by increases in fatty acid chain length and degree of unsaturation. Correlation studies suggested (a) an inverse relationship between lipoproteins (HDL and LDL+VLDL) and albumin, with a possible direct correlation to excreted (unassigned) pregnancy markers resonating at δ 0.55 and δ 0.63, (b) a direct link between LDL+VLDL and N-acetyl-glycoproteins, together with excreted marker at δ 0.55, and (c) correlation of plasma albumin with particular circulating and excreted metabolites. These results have unveiled specific lipoprotein/protein metabolic aspects of pregnancy with impact on the excreted metabolome and, therefore, provide an interesting lead for the further understanding of pregnancy metabolism.

Structural characterisation by MALDI-MS of olive xylo-oligosaccharides obtained by partial acid hydrolysis

Abstract Alkali extractable olive pulp and olive seed hull glucuronoxylans were submitted to mild acid hydrolysis originating low molecular weight xylo-oligosaccharides. The xylo-oligosaccharides thus formed were partially separated by size exclusion chromatography (Biogel P6) and several elution fractions were identified by MALDI-MS. The MALDI mass spectra obtained revealed the presence of several xylo-oligosaccharides ions in each fraction between the m / z ranges 500–2500. Based on the sugar and methylation analyses of the glucuronoxylans, the proposed structures are the neutral xylo-oligosaccharides (Xyl n , n =4–18) and acidic xylo-oligosaccharides substituted by glucuronic acid residues (Xyl n GlcA, n =3–16 and Xyl n GlcA 2 , n =2–14), by 4- O -methyl-glucuronic acid residues (Xyl n MeGlcA, n =3–14 and Xyl 2 MeGlcA 2 ) and by both acid residues (Xyl n GlcAMeGlcA, n =4–7). The identification of ions corresponding to long blocks of neutral and acidic oligosaccharides suggested that the acidic residues were irregularly distributed along the xylan backbone in both olive pulp and olive seed hull. In addition, MALDI mass spectra showed that the acidic xylo-oligosaccharides from the pulp were predominantly constituted by Xyl n MeGlcA oligosaccharides, while the acidic xylo-oligosaccharides from the seed hull were predominantly constituted by Xyl n GlcA and Xyl n GlcA 2 oligosaccharides.

Dietary curcumin counteracts extracellular transthyretin deposition: insights on the mechanism of amyloid inhibition.

The transthyretin amyloidoses (ATTR) are devastating diseases characterized by progressive neuropathy and/or cardiomyopathy for which novel therapeutic strategies are needed. We have recently shown that curcumin (diferuloylmethane), the major bioactive polyphenol of turmeric, strongly suppresses TTR fibril formation in vitro, either by stabilization of TTR tetramer or by generating nonfibrillar small intermediates that are innocuous to cultured neuronal cells. In the present study, we aim to assess the effect of curcumin on TTR amyloidogenesis in vivo, using a well characterized mouse model for familial amyloidotic polyneuropathy (FAP). Mice were given 2% (w/w) dietary curcumin or control diet for a six week period. Curcumin supplementation resulted in micromolar steady-state levels in plasma as determined by LC/MS/MS. We show that curcumin binds selectively to the TTR thyroxine-binding sites of the tetramer over all the other plasma proteins. The effect on plasma TTR stability was determined by isoelectric focusing (IEF) and curcumin was found to significantly increase TTR tetramer resistance to dissociation. Most importantly, immunohistochemistry (IHC) analysis of mice tissues demonstrated that curcumin reduced TTR load in as much as 70% and lowered cytotoxicity associated with TTR aggregation by decreasing activation of death receptor Fas/CD95, endoplasmic reticulum (ER) chaperone BiP and 3-nitrotyrosine in tissues. Taken together, our results highlight the potential use of curcumin as a lead molecule for the prevention and treatment of TTR amyloidosis.

Glycation and oxidation of histones H2B and H1: in vitro study and characterization by mass spectrometry

AbstractAmong the post-translational modifications, oxidation and glycation are of special interest, especially in diseases such as diabetes, and in aging. The synergistic interaction between glycation and oxidation, also known as “glycoxidation” is highly relevant due to its involvement in the production of deleterious changes at the molecular level. Non-enzymatic damage to nuclear proteins has potentially severe consequences for the maintenance of genomic integrity [54]. In this report, we study glycated histones and its in vitro oxidation. Data concerning the modifications that occurred in the histones were obtained by analysis of enzymatic digests (Glu-C and Arg-C) of unmodified and glycated histones, obtained before and after oxidation. Analysis was then performed using a MALDI-MS/MS-based approach combined with nano liquid chromatography. This approach allowed us to identify histone H2B and H1 specific-sites of oxidation and to distinguish the most affected residues for each histone. The results showed the occurrence of a cumulative effect of oxidative damage in the glycated histones when subjected to in vitro oxidation, suggesting that structural changes caused by glycation induces histones to a pro-oxidant state. Comparing the data of oxidized glycated histones with data from unmodified oxidized histones, using the same model of oxidation, the results clearly show that these oxidative modifications occur earlier and more extensively in glycated histones. Furthermore, the results pointed to an increased oxidative damage in the vicinity of the glycated residues.

Detection and Characterization by Mass Spectrometry of Radical Adducts Produced by Linoleic Acid Oxidation

The formation of linoleic acid radical species under the oxidative conditions of the Fenton reaction (using hydrogen peroxide and Fe (II)) was monitored by FAB-MS and ES-MS using the spin trap 5,5-dimethyl-1-pyrrolidine-N-oxide, DMPO. Both the FAB and ES mass spectra were very similar and showed the presence of ions corresponding to carbon- and oxygen centered spin adducts (DMPO/L·, DMPO/LO·, and DMPO/LOO·). Cyclic structures, formed between the DMPO oxygen and the neighboring carbon of the fatty acid, were also observed. Electrospray tandem mass spectrometry of these ions was performed to confirm the proposed structure of these adducts. All MS/MS spectra showed an ion at m/z 114, correspondent to the [DMPO+H]+, and a fragment ion due to loss of DMPO (loss of 113 Da), confirming that they are DMPO adducts. ES-MS/MS spectra of alkoxyl radical adducts (DMPO/LO·) showed an additional ion at m/z 130 [DMPO−O+H]+, while ES MS/MS of peroxyl radical adducts (DMPO/LOO·) showed a fragment ion at m/z 146 [DMPO−OO+H]+, confirming both structures. Other fragment ions were observed, such as alkyl acylium radical ions, formed by cleavage of the alkyl chain after loss of water and the DMPO molecule. The identification of fragment ions observed in the MS/MS spectra of the different DMPO adducts suggests the occurrence of structural isomers containing the DMPO moiety both at C9 and C13. The use of ES tandem mass spectrometry, associated with spin trapping experiments, has beee shown to be a valuable tool for the structural characterization of carbon and oxygen-centered spin adducts of lipid radicals.