Trichoderma spp. is one of the most commonly used biological control agents against plant pathogens. This fungus produces changes in plant metabolism, thus increasing growth and enhancing resistance to biotic and abiotic stresses. However, its modes of action remain to be defined. In the first hours of interaction between cucumber plant roots and Trichoderma asperellum strain T34, salicylic and jasmonic acid levels and typical antipathogenic peroxidase activity increase in the cotyledons to different degrees depending on the applied concentration of the fungi. The use of 2-DE protein profiling and MS analysis allowed us to identify 28 proteins whose expression was affected in cotyledons after cucumber root colonization by Trichoderma applied at high concentrations: 17 were found to be up-regulated while 11 were down-regulated. Proteins involved in ROS scavenging, stress response, isoprenoid and ethylene biosynthesis, and in photosynthesis, photorespiration, and carbohydrate metabolism were differentially regulated by Trichoderma. The proteome changes found in this study help to give an understanding of how Trichoderma-treated plants become more resistant to pathogen attacks through the changes in expression of a set of defence-oriented proteins which can directly protect the plant or switch the metabolism to a defensive, nonassimilatory state.

Imidazolidin-4-one derivatives of primaquine were synthesized as potential double prodrugs of the parent drug. The title compounds inhibit the development of the sporogonic cycle of Plasmodium berghei, affecting the appearance of oocysts in the midguts of the mosquitoes. The imidazolidin-4-ones are very stable, both in human plasma and in pH 7.4 buffer, indicating that they are active per se. Thus, imidazolidin-4-ones derived from 8-aminoquinolines represent a new entry in antimalarial structure-activity relationships.

Gilthead sea bream exposed to the cold show multiple physiological alterations, particularly in liver. A typical cold-stress response was reproduced in gilthead sea bream acclimated to 20 degrees C (Warm group) when the water temperature was lowered to 8 degrees C (Cold group). After 10 days, thiobarbituric acid reactive substances in the liver had increased by 50%, and nitric oxide had increased twofold. This indicates that lipid peroxidation and oxidative stress had occurred. Protein profiles of liver from fish in warm and cold environments were obtained by 2-DE. Quantification of differential expression by matching spots showed that a total of 57 proteins were altered significantly. Many proteins were downregulated following cold exposure, including actin, the most abundant protein in the proteome; enzymes of amino acid metabolism; and enzymes with antioxidant capacity, such as betaine-homocysteine-methyl transferase, glutathione-S-transferase and catalase. Some proteins associated with protective action were upregulated at low temperatures, including peroxiredoxin, thioredoxin and lysozyme; as well as enzymes such as aldehyde dehydrogenase and adenosin-methionine synthetase. However, the upregulation of proteases, proteasome activator protein and trypsinogen-like protein indicated an increase in proteolysis. Increases in elongation factor-1alpha, the GAPDH oxidative form, tubulin and Raf-kinase inhibitor protein indicated oxidative stress and the induction of apoptosis. These data indicate that cold exposure induced oxidative damage in hepatocytes.

No abstract

Escherichia coli Nissle 1917 (EcN) is a probiotic used for the treatment of intestinal disorders. EcN improves gastrointestinal homeostasis and microbiota balance; however, little is known about how this probiotic delivers effector molecules to the host. Outer membrane vesicles (OMVs) are constitutively produced by Gram-negative bacteria and have a relevant role in bacteria-host interactions. Using 1D SDS-PAGE and highly sensitive LC-MS/MS analysis we identified in this study 192 EcN vesicular proteins with high confidence in three independent biological replicates. Of these proteins, 18 were encoded by strain-linked genes and 57 were common to pathogen-derived OMVs. These proteins may contribute to the ability of this probiotic to colonize the human gut as they fulfil functions related to adhesion, immune modulation or bacterial survival in host niches. This study describes the first global OMV proteome of a probiotic strain and provides evidence that probiotic-derived OMVs contain proteins that can target these vesicles to the host and mediate their beneficial effects on intestinal function. All MS data have been deposited in the ProteomeXchange with identifier PXD000367 (http://proteomecentral.proteomexchange.org/dataset/PXD000367).

To screen one-bead-one-compound (OBOC) combinatorial libraries, tens of thousands to millions of compound beads are first mixed with a target molecule. The beads that interact with this molecule are then identified and isolated for compound structure determination. Here we describe an OBOC peptide library screening using streptavidin (SA) as probe protein, labeled with a red fluorescent dye and using the COPAS BIO-BEAD flow sorting equipment to separate fluorescent from nonfluorescent beads. The red dyes used were ATTO 590 and Texas Red. After incubating the library with the SA-red fluorescent dye conjugate, we isolated positive beads caused by peptide-SA interaction and false positive beads produced by peptide fluorescent dye interaction. These false positives were a drawback when sorting beads by COPAS. However,an in depth analysis of both kinds of beads allowed the differentiation of positives from false positives. The false positive beads showed bright homogeneous fluorescence, while positive beads had a heterogeneous fluorescence, exhibiting a characteristic halo appearance, with fluorescence intensity greatest at the bead surface and lowest in the core. The difference was more evident when using Texas Red instead of ATTO 590. Thus, positive beads could be manually separated from false positive ones. The beads were analyzed by matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS). Most of the sequences obtained from positive beads had the His-Pro-Gln motif. Peptides from false positive beads were rich in Leu/Ileu, His, Phe, and Tyr.

Pulmonary surfactant, a thin lipid/protein film lining mammalian lungs, functions in vivo to reduce the work of breathing and to prevent alveolar collapse. Analogues of two hydrophobic surfactant proteins, SP-B and SP-C, have been incorporated into therapeutic agents for respiratory distress syndrome, a pathological condition resulting from deficiency in surfactant. To facilitate rational design of therapeutic agents, a molecular level understanding of lipid interaction with surfactant proteins or their analogues in aqueous monolayer films is necessary. The current work uses infrared reflection-absorption spectroscopy (IRRAS) to determine peptide conformation and the effects of S-palmitoylation on the lipid interactions of a synthetic 13 residue N-terminal peptide [SP-C13(palm)(2)] of SP-C, in mixtures with 1,2-dipalmitoylphosphatidylcholine (DPPC) or 1,2-dipalmitoylphosphatidylglycerol (DPPG). Two Amide I' features, at approximately 1655 and approximately 1639 cm(-1) in the peptide IRRAS spectra, are assigned to alpha-helical peptide bonds in hydrophobic and aqueous environments, respectively. In binary DPPC/SP-C13(palm)(2) films, the proportion of hydrated/hydrophobic helix increases reversibly with surface pressure (pi), suggestive of the peptide being squeezed out from hydrophobic regions of the monolayer. No such effect was observed for DPPG/peptide monolayers, indicative of stronger, probably electrostatic, interactions. Depalmitoylation produced a weakened interaction with either phospholipid as deduced from IRRAS spectra and from pi-area isotherms. S-Palmitoylation may modulate peptide hydration and conformation in the N-terminal region of SP-C and may thus permit the peptide to remain in the film at the high surface pressures present during lung compression. The unique capability of IRRAS to detect the surface pressure dependence of protein or peptide structure/interactions in a physiologically relevant model for surfactant is clearly demonstrated.

Many Gram-negative, cold-adapted bacteria from the Antarctic environment produce large amounts of extracellular matter, which has potential biotechnology applications. We examined the ultrastructure of extracellular matter from five Antarctic bacteria (Shewanella livingstonensis NF22(T), Shewanella vesiculosa M7(T), Pseudoalteromonas sp. M4.2, Psychrobacter fozii NF23(T), and Marinobacter guineae M3B(T)) by transmission electron microscopy after high-pressure freezing and freeze substitution. All analyzed extracellular matter appeared as a netlike mesh composed of a capsular polymer around cells and large numbers of membrane vesicles (MVs), which have not yet been described for members of the genera Psychrobacter and Marinobacter. MVs showed the typical characteristics described for these structures, and seemed to be surrounded by the same capsular polymer as that found around the cells. The analysis of MV proteins from Antarctic strains by SDS-PAGE showed different banding profiles in MVs compared to the outer membrane, suggesting some kind of protein sorting during membrane vesicle formation. For the psychrotolerant bacterium, S. livingstonensis NF22(T), the growth temperature seemed to influence the amount and morphology of MVs. In an initial attempt to elucidate the functions of MVs for this psychrotolerant bacterium, we conducted a proteomic analysis on membrane vesicles from S. livingstonensis NF22(T) obtained at 4 and 18 degrees C. At both temperatures, MVs were highly enriched in outer membrane proteins and periplasmic proteins related to nutrient processing and transport in Gram-negative bacteria suggesting that MVs could be related with nutrient sensing and bacterial survival. Differences were observed in the expression of some proteins depending on incubation temperature but further studies will be necessary to define their roles and implications in the survival of bacteria in the extreme Antarctic environment.