Ana P. Barba de la Rosa

In vitro inhibition of dipeptidyl peptidase IV by peptides derived from the hydrolysis of amaranth (Amaranthus hypochondriacus L.) proteins

Bioactive compounds present in foods could potentially have beneficial effects on human health. In this study we report the in vitro inhibitory capacity of peptides released from amaranth seed proteins after enzymatic digestion, against dipeptidyl peptidase IV (DPPIV); an enzyme known to deactivate incretins, hormones involved in insulin secretion. Other seeds, such as soybean, black bean, and wheat were also tested. The highest inhibition of DPPIV was observed with amaranth peptides released after simulated gastrointestinal digestion, showing an IC(50) of 1.1mg/mL in a dose-dependent manner. In silico tryptic digestion of amaranth globulins was carried out releasing peptides larger than 13 residues. Some of these peptides were used for the in silico prediction of their binding modes with DPPIV. Docking models showed that the possible mechanism of globulin peptides to inhibit DPPIV was through blocking the active dimer formation. Peptides were also found inside the major cavity where the natural substrates reach the catalytic site of the enzyme. This is the first report of the identification of inhibitory DPPIV peptides from amaranth hydrolysates and the prediction of their binding modes at the molecular level, leading to their possible use as functional food ingredients in the prevention of diabetes.

Characterization of volatile compounds of mezcal, an ethnic alcoholic beverage obtained from Agave salmiana

Commercial mezcals (white, white with worm, rested, rested with worm, and aged) produced from Agave salmiana were analyzed by solid-phase microextraction-gas chromatography-mass spectrometry (SPME-GC-MS). Thirty-seven compounds were identified, and nine of them were classified as major compounds of mezcal (MCM). Saturated alcohols, ethyl acetate, ethyl 2-hydroxypropanoate, and acetic acid form the MCM group. Minor compounds of mezcal group include other alcohols, aldehydes, ketones, large chain ethyl esters, organic acids, furans, terpenes, alkenes, and alkynes. Most of the compounds found in mezcals in this study are similar to those present in tequilas and other alcoholic beverages. However, mezcals contain unique compounds such as limonene and pentyl butanoate, which can be used as markers for the authenticity of mezcal produced from A. salmiana.

Proteomic analysis of differentially accumulated proteins during ripening and in response to 1-MCP in papaya fruit

Papaya (Carica papaya L.) is a climacteric fruit susceptible to postharvest losses due to the ethylene-induced ripening. The inhibitor of ethylene action, 1-methylcyclopropene (1-MCP), has been used worldwide as a safe postharvest non-toxic agent, but the physiological and biochemical modifications induced by 1-MCP are not well understood. Using the 2-DE analysis, we report the changes in the protein profiles after 6 and 18 days of postharvest and the effect of the effect of 1-MCP treatment on fruits. Twenty seven protein spots showing differences in abundance during ripening were successfully identified by nano-LC-ESI/MS/MS. Some spots corresponded to the cell wall degrading enzymes related to fruit ripening; others were involved in oxidative damage protection, protein folding, and cell growth and survival that were induced by 1-MCP. This is the first proteomic report analyzing the effect of 1-MCP in papaya ripening. The present data will help to shed light on papaya fruit ripening process.

Amaranth lunasin-like peptide internalizes into the cell nucleus and inhibits chemical carcinogen-induced transformation of NIH-3T3 cells.

Because an unbalanced diet is an important risk factor for several illnesses, interest has increased in finding novel health-promoting foods. Amaranth produces seeds that not only have substantial nutritional properties but that also contain phytochemical compounds as rutin and nicotiflorin and peptides with antihypertensive and anticarcinogenic activities. We report that a cancer-preventive peptide in amaranth has activities similar to those of soybean lunasin. The amaranth lunasin-like peptide, however, requires less time than the soybean lunasin to internalize into the nucleus of NIH-3T3 cells, and inhibits histone acetylation (H(3) and H(4) in a 70 and 77%, respectively). The amaranth lunasin-like peptide inhibited the transformation of NIH-3T3 cells to cancerous foci. The open reading frame (ORF) of amaranth lunasin corresponds to a bifunctional inhibitor/lipid-transfer protein (LTP). LTPs are a family of proteins that in plants are implicated in different functions, albeit all linked to developmental processes and biotic and abiotic stress resistance. Our results open new intriguing questions about the function of lunasin in plants and support that amaranth is a food alternative containing natural peptides with health-promoting benefits.

Periplasmic expression and recovery of human interferon gamma in Escherichia coli

A synthetic human interferon gamma (hIFN-gamma) gene was fused to SP1 and SP3, two Sec-dependent artificial signal peptides to transport the hIFN-gamma to the periplasm of Escherichia coli BL21-SI. The processing efficiency of both SP1-hIFN-gamma and SP3-hIFN-gamma was dependent on the culture medium as well as the post-induction temperature. Both precursors were processed completely when cells were cultured using minimal medium and a post-induction temperature of 32.5 degrees C, and only the processed hIFN-gamma was detected. The SP3 signal peptide was more efficient than SP1 for the secretion of hIFN-gamma. Sixty percent of the total hIFN-gamma was secreted to the periplasm using the SP3 signal peptide and a post-induction temperature of 20 degrees C. Using Tris-sucrose-dithiothreitol (TSD) hypertonic buffer, the periplasmic soluble hINF-gamma was recovered with a purity of 85%.

Salt stress-induced alterations in the root proteome of Amaranthus cruentus L.

Salt stress is one of the major factors limiting crop productivity worldwide. Amaranth is a highly nutritious pseudocereal with remarkable nutraceutical properties; it is also a stress-tolerant plant, making it an alternative crop for sustainable food production in semiarid conditions. A two-dimensional electrophoresis gel coupled with a liquid chromatography-mass spectrometry/mass spectrometry (LC-MS/MS) approach was applied in order to analyze the changes in amaranth root protein accumulation in plants subjected to salt stress under hydroponic conditions during the osmotic phase (1 h), after recovery (24 h), and during the ionic phase of salt stress (168 h). A total of 101 protein spots were differentially accumulated in response to stress, in which 77 were successfully identified by LC-MS/MS and a database search against public and amaranth transcriptome databases. The resulting proteins were grouped into different categories of biological processes according to Gene Ontology. The identification of several protein isoforms with a change in pI and/or molecular weight reveals the importance of the salt-stress-induced posttranslational modifications in stress tolerance. Interestingly stress-responsive proteins unique to amaranth, for example, Ah24, were identified. Amaranth is a stress-tolerant alternative crop for sustainable food production, and the understanding of amaranths stress tolerance mechanisms will provide valuable input to improve stress tolerance of other crop plants.

Characterization of Antidiabetic and Antihypertensive Properties of Canary Seed (Phalaris canariensis L.) Peptides

Canary grass is used as traditional food for diabetes and hypertension treatment. The aim of this work is to characterize the biological activity of encrypted peptides released after gastrointestinal digestion of canary seed proteins. Canary peptides showed 43.5% inhibition of dipeptidyl peptidase IV (DPPIV) and 73.5% inhibition of angiotensin-converting enzyme (ACE) activity. An isolated perfused rat heart system was used to evaluate the canary seed vasoactive effect. Nitric oxide (NO), a major vasodilator agent, was evaluated in the venous effluent from isolated perfused rat heart. Canary seed peptides (1 μg/mL) were able to induce the production of NO (12.24 μM) in amounts similar to those induced by captopril (CPT) and bradykinin (BK). These results show that encrypted peptides in canary seed have inhibitory activity against DPPIV and ACE, enzymes that are targets for diabetes and hypertension treatments.

Crystal structure of a major seed storage protein, 11S proglobulin, from Amaranthus hypochondriacus: Insight into its physico-chemical properties

Amaranth is a crop known for its high quality proteins. 11S Globulin is one of the most abundant and important storage proteins of the amaranth grain. Here, we report the crystal structure of amaranth 11S proglobulin at a final resolution of 2.28 Å. It belonged to the space group P6(3) with cell dimensions a=b=96.6, c=75.0 Å. It contains one asymmetric unit consisting of 372 residues and 100 water molecules. Disordered regions in the model approximately correspond to the variable regions of the 11S globulins. The structure has an extended α-helix and β-barrel domains at both N-terminal and C-terminal regions, which are characteristic of the 11S and 7S globulins. The three dimensional structure suggests that its high thermal stability is due to the cumulative effects of many factors and its good emulsifying property depended on the balance between its surface hydrophobicity and hydrophilicity.

Synthesis and quantitation of six phenolic amides in Amaranthus spp.

Cinnamoylphenethylamines are phenolic amides in which cinnamic acid provides the acid moiety and phenethylamine the amine moiety. Single ion monitoring (SIM) in LC-MS was performed on amaranth leaf extracts. Masses corresponding to sets of regioisomers, including previously reported compounds, were examined. Six peaks were detected and their corresponding standards synthesized for a quantitative LC-MS/MS investigation of cinnamoylphenethylamines in amaranth. Four cinnamoylphenethylamines (caffeoyltyramine, feruloyldopamine, sinapoyltyramine, and p-coumaroyltyramine) are reported in the Amaranthaceae for the first time; also, one rare compound, feruloyl-4-O-methyldopamine, appeared to be quite common in the genus Amaranthus. Feruloyldopamine showed moderate antifungal activity toward an isolate of Fusarium culmorum. Our LC-MS approach, in conjunction with the straightforward synthesis, provides a simple, reliable way of quantitatively investigating cinnamoylphenethylamines in plants. Concentrations of cinnamoylphenethylamines vary widely: feruloyltyramine was present in quantities of 5.26 to 114.31 microg/g and feruloyldopamine in quantities of 0.16 to 10.27 microg/g, depending on the plant sample.

Proteomic Analysis of Amaranth (Amaranthus hypochondriacus L.) Leaves under Drought Stress

Amaranth (Amaranthus hypochondriacus L.) is a plant that produces seed with high protein content, is rich on nutraceutical compounds, and can grow under environmental conditions where most of the basic crops are not able to develop. But little is know about the amaranth stress‐responsive genes/proteins. The aim of this work was to apply the comparative proteomics approach to study the differential expression of amaranth leaf proteins under drought stress. However, the protein extraction from amaranth tissues is difficult as a result of high endogenous concentrations of interfering compounds; we have made some modifications of the classical trichloroacetic acid–acetone precipitation method to improve the quantity and quality of extracted proteins. Satisfactory and reproducible two‐dimensional electrophoresis protein profiles were obtained; the method was also tested forAgave tequilana and Opuntia spp., two more examples of plants that are tolerant to drought stress. Drought‐responsive proteins in amaranth leaves were identified by liquid chromatography electrospray ionization tandem mass spectrometry (LC/ESI‐MS/MS). The upregulated proteins identified included chloroplast chaperonins involved in refolding and protein complexes protection. Downregulated proteins include Rubisco large subunit, cytochrome b6f, oxygen evolving complexes, and the ascorbate peroxidase mitochondrial. The results have shown that chloroplasts and mitochondria may play a central role in amaranth adaptation to abiotic stress, and further studies should be done at the subcellular level.