Psd2 pea defensin shows a preference for mimetic membrane rafts enriched with glucosylceramide and ergosterol.

Psd2 is a pea defensin with 47 amino acid residues that inhibits the growth of fungal species by an uncharacterized mechanism. In this work, Psd2 interactions with model membranes mimicking the lipid compositions of different organisms were evaluated. Protein-lipid overlay assays indicated that Psd2 recognizes Fusarium solani glucosylceramide (GlcCerF.solani) and ergosterol (Erg) in addition to phosphatidylcholine (POPC) and some phosphatidylinositol species, such as PtdIns (3)P, (5)P and (3,5)P2, suggesting that these lipids may play important roles as Psd2 targets. Assays using lipid vesicles were also performed to study the behaviour and dynamics that occur after peptide-membrane interactions. Surface plasmon resonance analysis showed that Psd2 has a higher affinity for pure POPC and POPC-based vesicles containing GlcCer and Erg at a 70:30 proportion than for vesicles containing cholesterol (Chol). Partition experiments by fluorescence spectroscopy showed a decrease in Trp42 quantum yield of Psd2 in the presence of GlcCerF.solani and Erg, individually or in simultaneously enriched membranes. The partition coefficient (Kp) obtained indicated a Psd2 partition preference for this vesicles, confirmed by quenching assays using acrylamide and 5/16-doxyl-stearic acid. Furthermore, we showed that the presence of C8C9 double bonds and a methyl group at position C9 of the sphingoid base backbone of GlcCer was relevant to Psd2 activity against Aspergillus nidulans. These results are consistent with the selectivity of Psd2 against fungi and its lack of toxicity in human erythrocytes. Psd2 represents a promising natural compound for the treatment of fungal infections.

Plasmodium falciparum invasion and intraerythrocytic development are impaired by 2', 3'-dialdehyde adenosine.

Purine nucleotide synthesis in protozoa takes place exclusively via the purine salvage pathway and S-adenosyl-l-homocysteine hydrolase (SAHH) is an important enzyme in the Plasmodium salvage pathway which is not present in erythrocytes. Here, we describe the antimalarial effect of 2'3'-dialdehyde adenosine or oxidized adenosine (oADO), inhibitor of SAHH, on in vitro infection of human erythrocytes by P. falciparum. Treatment of infected erythrocytes with oADO inhibits parasite development and reinvasion of new cells. Erythrocytes pre-treated with oADO have a reduced susceptibility to invasion. Our results suggest that oADO interferes with one or more parasitic enzymes of the purine salvage pathway.

Structural changes of fibrinogen molecule mediated by the N-homocysteinylation reaction.

Homocysteine and its cyclic ester homocysteine thiolactone (HTL) have been involved in the detrimental consequences associated to hyperhomocysteinemia, an independent risk factor for vascular diseases. HTL reacts with protein lysine residues in a process named N-homocysteinylation. The aim of our study was to evaluate the in vitro effects of HTL on the fibrinogen through electrophoretic methods. Fibrinogen was incubated with HTL at different molar ratios and structural changes of the protein were assessed by polyacrylamide gel electrophoresis (PAGE), capillary zone electrophoresis (CZE) and capillary isoelectric focusing (CIEF). Ellman´s reaction, CZE and proton nuclear magnetic resonance (1H NMR) were used to evaluate HTL hydrolyisis. On denaturing PAGE numerous bands were observed, being the three lower bands identical to those obtained by treatment with 2-mercaptoethanol. This effect was also detected by CZE. The results show a reducing action of HTL on the fibrinogen molecule, probably attributed to the sulfhydryl groups generated by N-homocysteinylation and/or by the ones present in the homocysteine molecule yielded by HTL hydrolysis. In order to distinguish between these two options, HTL stability was evaluated at different pH and incubation times. The results showed minimum HTL hydrolysis in our experimental conditions. We postulate that the reducing effect observed would be mainly associated to the new sulfhydryl groups generated by the N-homocysteinylation process. Moreover, a displacement of the HTL-treated fibrinogen isoforms towards more acidic pH values was detected. The structural changes of N-homocysteinylated fibrinogen could be involved in the pathological consequences of hyperhomocysteinemia.

Lipase-catalyzed regioselective preparation of fatty acid esters of hydrocortisone.

A series of fatty acid derivatives of hydrocortisone has been prepared by an enzymatic methodology. Nine 21-monoacyl products and one 3,11,17-triacetyl derivative, nine of them novel compounds, were obtained in a highly regioselective way through lipase-catalyzed esterification, transesterification and alcoholysis reactions. The influence of various reaction parameters such as acylating agent: substrate ratio, enzyme: substrate ratio, solvent, temperature and nature of acylating agent and alcohol was evaluated. Among the tested lipases, Candida antarctica lipase appeared to be the most appropriate and showed a high efficient behavior especially in a one-pot transesterification. The advantages presented by this methodology, such as mild reaction conditions and low environmental impact, make the biocatalysis a convenient way to prepare acyl derivatives of hydrocortisone. These lipophilic compounds are potential products in the pharmaceutical industry.