Size matters: TLR4-mediated effects of α-(1,5)-linear arabino-oligosaccharides in macrophage-like cells depend on their degree of polymerization.

Linear arabino-oligosaccharides (LAOS) produced from controlled enzymatic hydrolysis of arabinans from sugar beet are well-known because of their chain-length dependent prebiotic effects. However, it is not clear if these α-(1,5)-linked arabinose oligosaccharides can interact directly with immune system cells, as well as if its degree of polymerization (DP) influences possible biological effects. Four high purity LAOS with distinct DP were tested in macrophage-like cells exposed or not to LPS. Results shown that LAOS interact with Toll-like receptor (TLR) 4 in a chain length-dependent manner. LAOS with higher DP induce stimulatory effects mainly through the TLR4/MyD88 pathway, thereby enhancing the release of tumor necrosis factor alpha (TNF-α), interleukin (IL-) 1ß, 6, 12, and chemokines including MCP-1, RANTES, IL-8, and IP-10. Notably, LAOS with lower DP appears to have an opposite effect to those counterparts with higher DP, as they does not induce the secretion of cytokines and chemokines in macrophages-like cells, while also inhibit TLR4-mediated effects induced by both lipopolysaccharide and LAOS with higher DP. These findings provide not only insights into potential biological effects of LAOS, but also reveal that controlled enzymatic hydrolysis of sugar beet arabinans may lead to dietary oligosaccharides with desired biological properties.

The Starch Is (Not) Just Another Brick in the Wall: The Primary Metabolism of Sugars During Banana Ripening.

The monocot banana fruit is one of the most important crops worldwide. As a typical climacteric fruit, the harvest of commercial bananas usually occurs when the fruit is physiologically mature but unripe. The universal treatment of green bananas with ethylene or ethylene-releasing compounds in order to accelerate and standardize the ripening of a bunch of bananas mimics natural maturation after increasing the exogenous production of ethylene. The trigger of autocatalytic ethylene production regulated by a dual positive feedback loop circuit derived from a NAC gene and three MADS genes results in metabolic processes that induce changes in the primary metabolism of bananas. These changes include pulp softening and sweetening which are sensorial attributes that determine banana postharvest quality. During fruit development, bananas accumulate large amounts of starch (between 15 and 35% w/w of their fresh weight, depending on the cultivar). Pulp softening and sweetening during banana ripening are attributed not only to changes in the activities of cell wall hydrolases but also to starch-to-sugar metabolism. Therefore, starch granule erosion and disassembling are key events that lead bananas to reach their optimal postharvest quality. The knowledge of the mechanisms that regulate sugar primary metabolism during banana ripening is fundamental to reduce postharvest losses and improve final product quality, though. Recent studies have shown that ethylene-mediated regulation of starch-degrading enzymes at transcriptional and translational levels is crucial for sugar metabolism in banana ripening. Furthermore, the crosstalk between ethylene and other hormones including indole-3-acetic acid and abscisic acid also influences primary sugar metabolism. In this review, we will describe the state-of-the-art sugar primary metabolism in bananas and discuss the recent findings that shed light on the understanding of the molecular mechanisms involved in the regulation of this metabolism during fruit ripening.

Polysaccharides from chayote enhance lipid efflux and regulate NLRP3 inflammasome priming in macrophage-like THP-1 cells exposed to cholesterol crystals.

The contribution of dietary fiber to decrease the risk of atherosclerosis may occur through other mechanisms besides the increased excretion of cholesterol. Although macrophages are crucial for lipid clearance, the excessive uptake of cholesterol crystals (CC) by these cells induce NLRP3 inflammasome and foam cell formation. Thus, we investigated whether the water-soluble DF from chayote (WSP) regulate CC-pretreated macrophage-like THP-1 cells. Linkage analysis indicated that WSP is composed mainly of pectic homogalacturonan and highly branched type I rhamnogalacturonan as well as hemicellulosic material including glucomannan, xyloglucan, and glucurono(arabino)xylan. WSP reduced interleukin (IL)-1ß and chemokine release in CC-pretreated macrophages. Notably, WSP also reduced lipid accumulation in cells previously exposed to CC. Furthermore, WSP upregulated liver X receptor alpha expression, which may account for increased lipid efflux, and reduced matrix metallopeptidase 9 expression. WSP also reduced active caspase-1 protein levels, and downregulated NLRP3 and IL-1ß gene expression in CC-pretreated cells, suggesting that this polysaccharide fraction regulates the priming signals required for NLRP3 inflammasome activation. Thus, WSP regulate lipid efflux and suppress inflammasome priming in macrophages, suggesting that the health benefits of this dietary fiber could go beyond its physical properties on the gastrointestinal tract.

α- and ß-d-Glucans from the edible mushroom Pleurotus albidus differentially regulate lipid-induced inflammation and foam cell formation in human macrophage-like THP-1 cells.

Macrophages play an essential role in lipid metabolism; however, the excessive uptake of modified lipids and cholesterol crystals (CC) leads to the formation of pro-inflammatory lipid-laden macrophages called foam cells. Since the α-1,6- and ß-1,3-d-glucans from the basidiome and the mycelium of the edible mushroom Pleurotus albidus have previously been shown to regulate macrophage function, these glucans were tested in macrophage-like THP-1 cells previously exposed to acetylated low-density lipoproteins (acLDL) or CC. The glucans inhibited lipid-induced inflammation, but only the ß-1,3-d-glucan regulated both the NLRP3 inflammasome activation and the expression of genes involved on lipid efflux in acLDL- or CC-pretreated cells, thereby reducing foam cell formation. In contrast, the two α-1,6-glucans tested inhibited foam cell formation only in acLDL-pretreated cells and had no effect on the expression of the peroxisome proliferator-activated receptor gamma and liver X receptor alpha genes, suggesting that these glucans regulate lipid influx rather than lipid efflux. Thus, α- and ß-d-glucans differentially regulate lipid-induced inflammation and foam cell formation in macrophage-like cells. Furthermore, results emphasize that P. albidus has potential to be used as a functional food or as a source for the extraction of biologically-active glucans.

Characterization and immunomodulatory effects of glucans from Pleurotus albidus, a promising species of mushroom for farming and biomass production.

Polysaccharides from a number of mushroom species are recognized as functional food ingredients with potential health benefits, including immunomodulatory effects. In this study, polysaccharides extracted from the basidiome with cold water (BaCW), hot water (BaHW), and hot alkali (BaHA) solution, and exo- (MyEX) and endopolysaccharides (MyEN) from the submerged culture of Pleurotus albidus, a promising species for farming and biomass production, were analyzed for their chemical composition and structure and immunomodulatory effects on macrophages. Compositional (HPAEC-PAD and HPSEC-RID/MWD) and structural (FT-IR, 1D- and 2D-NMR) analyses identified BaCW and MyEX as ß-(1,6)-branched ß-(1,3)-glucans, BaHW and MyEN as α-(1,3)-(1,2)-branched α-(1,6)-glucans, and BaHA as a mixture of α-(1,6)- and ß-(1,3)-glucans. BaCW and MyEX stimulated the production of tumor necrosis factor alpha (TNF-α) and nitric oxide (NO), but not interleukin-6 (IL-6), and decreased phagocytosis of zymosan particles. In contrast, BaHW and MyEN induced TNF-α, NO and IL-6 production, and increased zymosan phagocytosis, while BaHA displayed intermediary effects in comparison the other polysaccharides. In conclusion, the basidiome and the submerged culture of P. albidus are sources of easily extractable α- and ß-glucans with potential immunomodulatory effects.

Comparative Proteome Analysis of the Tuberous Roots of Six Cassava (Manihot esculenta) Varieties Reveals Proteins Related to Phenotypic Traits.

Cassava (Manihot esculenta Crantz) is a staple food and an important source of starch, and the attributes of its tuberous root largely depend on the variety. The proteome of cassava has been investigated; however, to date, no study has focused on varieties that reveal the molecular basis of phenotypical characteristics. Therefore, we aimed to compare the proteome of the tuberous roots of six cassava varieties that differed in carbohydrates, carotenoids, and resistance to diseases, among other attributes. Two-dimensional gels showed 146 differential spots between the varieties, and the functional roles of some differential proteins were correlated to phenotypic characteristics of the varieties, such as the amount of carbohydrates or carotenoids and the resistance to biotic or abiotic stresses. The results obtained here highlight elements that might help to direct the improvement of new cultivars of cassava, which is an economically and socially relevant crop worldwide.

The water-soluble non-starch polysaccharides from bananas display immunomodulatory properties on cultured macrophages.

Some diet components, such as certain indigestible polysaccharides from edible plants, may interact with the gut-associated lymphoid tissue and improve the host immune response to pathogens. The non-starch polysaccharides (NSP) from bananas are non-digestible carbohydrates that resemble some immunomodulatory polysaccharides occurring in the cell wall of cereals. Based on this similarity, the effects of the water-soluble NSP from two banana cultivars (Nanicão and Thap Maeo) on the phagocytic activity, nitric oxide (NO) and cytokines produced by cultured macrophages were investigated. An investigation into the monosaccharide composition and the oligomers released by enzymatic hydrolysis of the ultra-filtered fraction above 50kDa and the heat-treated fraction of water-soluble NSP from both cultivars revealed they are mostly composed of mannan and galacturonans (homogalacturonan, xylogalacturonan and rhamnogalacturonan). The NSP tested were able to activate the macrophages, but the effects on the phagocytic activity and the release of tumor necrosis factor alpha (TNF-α), interleukin-6 (IL-6) and nitric oxide production were dependent on the polysaccharide concentration and the cultivar origin. Despite some specific differences, the NSP from Nanicão and Thap Maeo banana fruits may be considered prospective food immunomodulators, which contribute to the promotion of a more responsive immune system.

Analysis of papaya cell wall-related genes during fruit ripening indicates a central role of polygalacturonases during pulp softening.

Papaya (Carica papaya L.) is a climacteric fleshy fruit that undergoes dramatic changes during ripening, most noticeably a severe pulp softening. However, little is known regarding the genetics of the cell wall metabolism in papayas. The present work describes the identification and characterization of genes related to pulp softening. We used gene expression profiling to analyze the correlations and co-expression networks of cell wall-related genes, and the results suggest that papaya pulp softening is accomplished by the interactions of multiple glycoside hydrolases. The polygalacturonase cpPG1 appeared to play a central role in the network and was further studied. The transient expression of cpPG1 in papaya results in pulp softening and leaf necrosis in the absence of ethylene action and confirms its role in papaya fruit ripening.

2D-DIGE analysis of mango (Mangifera indica L.) fruit reveals major proteomic changes associated with ripening.

A comparative proteomic investigation between the pre-climacteric and climacteric mango fruits (cv. Keitt) was performed to identify protein species with variable abundance during ripening. Proteins were phenol-extracted from fruits, cyanine-dye-labeled, and separated on 2D gels at pH 4-7. Total spot count of about 373 proteins spots was detected in each gel and forty-seven were consistently different between pre-climacteric and climacteric fruits and were subjected to LC-MS/MS analysis. Functional classification revealed that protein species involved in carbon fixation and hormone biosynthesis decreased during ripening, whereas those related to catabolism and the stress-response, including oxidative stress and abiotic and pathogen defense factors, accumulated. In relation to fruit quality, protein species putatively involved in color development and pulp softening were also identified. This study on mango proteomics provides an overview of the biological processes that occur during ripening.

Molecular cloning and characterization of a ripening-induced polygalacturonase related to papaya fruit softening.

Pulp softening is one of the most remarkable changes during ripening of papaya (Carica papaya) fruit and it is a major cause for post-harvest losses. Although cell wall catabolism has a major influence on papaya fruit, quality information on the gene products involved in this process is limited. A full-length polygalacturonase cDNA (cpPG) was isolated from papaya pulp and used to study gene expression and enzyme activity during normal and ethylene-induced ripening and after exposure of the fruit to 1-MCP. Northern-blot analysis demonstrated that cpPG transcription was strongly induced during ripening and was highly ethylene-dependent. The accumulation of cpPG transcript was paralleled by enzyme activity, and inversely correlated to the pulp firmness. Preliminary in silico analysis of the cpPG genomic sequence revealed the occurrence of putative regulatory motifs in the promoter region that may help to explain the effects of plant hormones and non-abiotic stresses on papaya fruit firmness. This newly isolated cpPG is an important candidate for functional characterization and manipulation to control the process of pulp softening during papaya ripening.

Changes in cell wall composition associated to the softening of ripening papaya: evidence of extensive solubilization of large molecular mass galactouronides.

Papaya (Carica papaya) is a climacteric fruit that undergoes dramatic pulp softening. Fruits sampled at three different conditions (natural ripening or after exposition to ethylene or 1-methylcyclopropene) were used for the isolation of cell wall polymers to find changes in their degradation pattern. Polymers were separated according to their solubility in water, CDTA, and 4 M alkali, and their monosaccharide compositions were determined. Water-soluble polymers were further characterized, and their increased yields in control and ethylene-treated fruit, in contrast to those that were treated with 1-MCP, indicated a strong association between fruit softening and changes in the cell wall water-soluble polysaccharide fraction. The results indicate that the extensive softening in the pulp of ripening papayas is a consequence of solubilization of large molecular mass galacturonans from the pectin fraction of the cell wall. This process seems to be dependent on the levels of ethylene, and it is likely that the releasing of galacturonan chains results from an endo acting polygalacturonase.

Mango starch degradation. II. The binding of alpha-amylase and beta-amylase to the starch granule.

During mango ripening, soluble sugars that account for mango sweetening are accumulated through carbon supplied by both photosynthesis and starch degradation. The cultivar Keitt has a characteristic dependence on sugar accumulation during starch degradation, which takes place during ripening, only a few days after detachment from the tree. Most knowledge about starch degradation is based on seeds and leaves currently used as models. However, information about the mango fruit is scarce. This work presents the evaluation of alpha- and beta-amylases in the starch granule surface during fruit development and ripening. Extractable proteins were assayed for amylase activity and detected by immunofluorescence microscopy and correlated to gene expression. The results suggest that both amylases are involved in starch degradation during mango ripening, probably under the dependence of another signal triggered by the detachment from the mother-plant.

Mango starch degradation. I. A microscopic view of the granule during ripening.

The starch content of unripe mango Keitt is around 7% (FW), and it is converted to soluble sugars during the ripening of the detached fruit. Despite the importance of starch-to-soluble sugar metabolism for mango quality, little literature is found on this subject and none concerning the physical aspects of starch degradation. This manuscript presents some changes in the physical aspects of the starch granule during ripening, as analyzed by light microscopy, scanning electron microscopy (SEM), and atomic force microscopy (AFM). According to the analysis, unripe Keitt-mango-starch being spherical in shape and measuring around 15 microm, has A-type X-ray diffraction pattern with a degree of crystallinity around 21% with slight changes after 8 days of ripening. AFM images of the surface of the granules showed ultra microstructures, which are in agreement with a blocklet-based organization of the granules. The AFM-contrast image of growing layers covering the granule showed fibril-like structures, having 20 nm in diameter, transversally connecting the layer to the granule. The appearance of the partially degraded granules and the pattern of degradation were similar to those observed as a result of amylase activity, suggesting a hydrolytic pathway for the degradation of starch from mango cultivar Keitt. These results provide clues to a better understanding of starch degradation in fruits.

Molecular cloning and characterization of an alpha-amylase occurring in the pulp of ripening bananas and its expression in Pichia pastoris.

Alpha-amylases (EC 3.2.1.1) are glycosyl hydrolases with endoglycolytic activity on the alpha-1,4-d-glucosidic linkages in starch. In bananas, the mobilization of starch accounts for sugar accumulation during ripening, and among several hydrolytic enzymes, alpha-amylase is the only enzyme argued to be able to attack the intact granules, indicating a pivotal role for this enzyme. A 1953 bp full-length banana alpha-amylase cDNA (MAmy), encoded for a sequence of 416 amino acids, was cloned and used for heterologous expression in Pichia pastoris. The cloned MAmy presented the highly conserved motifs common to alpha-amylases, and the amylolytic activity of the extracts from yeast transformed with MAmy demonstrated that it encodes for a functional alpha-amylase, suggesting a putative role for this gene in starch degradation during fruit ripening.