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The physiological functions of sphingolipids in animals have been intensively studied, while less attention has been paid to their roles in plants. Here, we reveal the involvement of sphingolipid delta8 desaturase (SlSLD) in the chilling resistance of tomato (Solanum lycopersicum cv. Micro-Tom). We used the virus-induced gene silencing (VIGS) approach to knock-down SlSLD expression in tomato leaves, and then evaluated chilling resistance. Changes in leaf cell structure under a chilling treatment were observed by transmission electron microscopy. In control plants, SlSLD was highly expressed in the fruit and leaves in response to a chilling treatment. The degree of chilling damage was greater in SlSLD-silenced plants than in control plants, indicating that SlSLD knock-down significantly reduced the chilling resistance of tomato. Compared with control plants, SlSLD-silenced plants showed higher relative electrolytic leakage and malondialdehyde content, and lower superoxide dismutase and peroxidase activities after a chilling treatment. Chilling severely damaged the chloroplasts in SlSLD-silenced plants, resulting in the disruption of chloroplast membranes, swelling of thylakoids, and reduced granal stacking. Together, these results show that SlSLD is crucial for chilling resistance in tomato.
Gut bacteria exert beneficial and harmful effects in metabolic diseases as deduced from the comparison of germfree and conventional mice and from fecal transplantation studies. Compositional microbial changes in diseased subjects have been linked to adiposity, type 2 diabetes and dyslipidemia. Promotion of an increased expression of intestinal nutrient transporters or a modified lipid and bile acid metabolism by the intestinal microbiota could result in an increased nutrient absorption by the host. The degradation of dietary fiber and the subsequent fermentation of monosaccharides to short-chain fatty acids (SCFA) is one of the most controversially discussed mechanisms of how gut bacteria impact host physiology. Fibers reduce the energy density of the diet, and the resulting SCFA promote intestinal gluconeogenesis, incretin formation and subsequently satiety. However, SCFA also deliver energy to the host and support liponeogenesis. Thus far, there is little knowledge on bacterial species that promote or prevent metabolic disease. Clostridium ramosum and Enterococcus cloacae were demonstrated to promote obesity in gnotobiotic mouse models, whereas bifidobacteria and Akkermansia muciniphila were associated with favorable phenotypes in conventional mice, especially when oligofructose was fed. How diet modulates the gut microbiota towards a beneficial or harmful composition needs further research. Gnotobiotic animals are a valuable tool to elucidate mechanisms underlying diet-host-microbe interactions.
The whey protein beta-lactoglobulin has been proposed as a transporter for covalent bound bioactive compounds in order to enhance their stability and reduce their sensory perception. The garlic derived compounds allicin and diallyl disulfide were bound covalently to the native and heat denatured protein. The binding site and the influence of the modification on the digestibility were determined by mass spectrometric analysis of the modified beta-lactoglobulin. Further, the conformation of the modified protein was assessed by circular dichroism and dynamic light scattering. The free thiol group of Cys(121) turned out to be the major binding site. After proteolysis with trypsin at pH 7 but not with pepsin at pH 2, a limited transfer to other cysteinyl residues was observed. The covalently bound ligands did not mask any proteolytic cleavage sites of pepsin, trypsin or chymotrypsin. The modified beta-lactoglobulin showed a native like conformation, besides a moderate loosening of protein folding. The covalent binding of organosulfur compounds to beta-lactoglobulin provides a bioactive ingredient without impairing the digestibility and functional properties of the protein. (C) 2015 Elsevier Ltd. All rights reserved.
Identification and LC-MS/MS-based analyses of technical enzymes in wheat flour and baked products
(2016)
The use of technical enzymes in bakery industry is necessary for a consistent and good quality of baked products. Since the cultivation of cereals leads to low amounts of endogenous enzymes being present, a need of their commercial alternatives is becoming a routine process in order to meet the consumer quality demands. Targeted quantification proteomics-based methods are necessary for their detection to meet the regulatory criteria. Here, we initially report on the identification of Lipase FE-01, a lipase from fungus Thermomyces lanuginosus, as analyzed by SDS-PAGE, in-Gel digestion, and MALDI-TOF-MS. In further experiments, the focus of the study was directed toward an extensive use and optimization of in-solution enzymatic digestion in combination with LC-MS/MS techniques in identification of specific peptide markers and finally in utilization of the latter in delivering reproducible quantification data for several different technical enzymes (alpha-amylases, xylanase, and lipases from microbial origin) in complex matrices such as baked bread and wheat flour. Two digestion protocols (a fast option using thermocycler program and the well-established overnight method) were tested, and both of these can be successfully applied. The application of isotopically labeled analogs of the MRM targeted peptides as internal standards and the addition of an internal protein standard during the extraction/digestion experiment were compared to determine the optimal quantification algorithm of the recovered enzyme concentrations. Thus, a standardized sensitive LC-MS/MS method could be developed to determine technical enzymes as forthcoming ingredients in the prefabricated food formulations in concentrations lower than 10 ppm.
Dipeptidyl peptidase (DPP)-4 inhibitors delay chronic kidney disease (CKD) progression in experimental diabetic nephropathy in a glucose-independent manner. Here we compared the effects of the DPP-4 inhibitor linagliptin versus telmisartan in preventing CKD progression in non-diabetic rats with 5/6 nephrectomy. Animals were allocated to 1 of 4 groups: sham operated plus placebo; 5/6 nephrectomy plus placebo; 5/6 nephrectomy plus linagliptin; and 5/6 nephrectomy plus telmisartan. Interstitial fibrosis was significantly decreased by 48% with linagliptin but a non-significant 24% with telmisartan versus placebo. The urine albumin-to-creatinine ratio was significantly decreased by 66% with linagliptin and 92% with telmisartan versus placebo. Blood pressure was significantly lowered by telmisartan, but it was not affected by linagliptin. As shown by mass spectrometry, the number of altered peptide signals for linagliptin in plasma was 552 and 320 in the kidney. For telmisartan, there were 108 peptide changes in plasma and 363 in the kidney versus placebo. Linagliptin up-regulated peptides derived from collagen type I, apolipoprotein C1, and heterogeneous nuclear ribonucleoproteins A2/B1, a potential downstream target of atrial natriuretic peptide, whereas telmisartan up-regulated angiotensin II. A second study was conducted to confirm these findings in 5/6 nephrectomy wild-type and genetically deficient DPP-4 rats treated with linagliptin or placebo. Linagliptin therapy in wild-type rats was as effective as DPP-4 genetic deficiency in terms of albuminuria reduction. Thus, linagliptin showed comparable efficacy to telmisartan in preventing CKD progression in non-diabetic rats with 5/6 nephrectomy. However, the underlying pathways seem to be different. Copyright (C) 2016, International Society of Nephrology. Published by Elsevier Inc. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).
Sections of fecal cylinders were analyzed using fluorescence in situ hybridization targeting 180 bacterial groups. Samples were collected from three groups of women (N = 20 each) treated for bacterial vaginosis with ciprofloxacin + metronidazole. Group A only received the combined antibiotic regimen, whereas the A/Sb group received concomitant Saccharomyces boulardii CNCM I-745 treatment, and the A.Sb group received S. boulardii prophylaxis following the 14-day antibiotic course. The number of stool cylinders analyzed was 188 out of 228 in group A, 170 out of 228 in group A/Sb, and 172 out of 216 in group Ash. The colonic biomass was organized into a separate mucus layer with no bacteria, a 10-30 mu m broad unstirred transitional layer enriched with bacteria, and a patchy fermentative area that mixed digestive leftovers with bacteria. The antibiotics suppressed bacteria mainly in the fermentative area, whereas abundant bacterial clades retreated to the transitional mucus and survived. As a result, the total concentration of bacteria decreased only by one order. These effects were lasting, since the overall recovery of the microbial mass, bacterial diversity and concentrations were still below pre-antibiotic values 4 months after the end of antibiotic treatment. Sb-prophylaxis markedly reduced antibiotic effects and improved the recovery rates. Since the colon is a sophisticated bioreactor, the study indicated that the spatial anatomy of its biomass was crucial for its function. (C) 2015 The Authors. Published by Elsevier GmbH. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/).
Plasma carotenoids, tocopherols, and retinol in the age-stratified (35–74 years) general population
(2016)
Blood micronutrient status may change with age. We analyzed plasma carotenoids, α-/γ-tocopherol, and retinol and their associations with age, demographic characteristics, and dietary habits (assessed by a short food frequency questionnaire) in a cross-sectional study of 2118 women and men (age-stratified from 35 to 74 years) of the general population from six European countries. Higher age was associated with lower lycopene and α-/β-carotene and higher β-cryptoxanthin, lutein, zeaxanthin, α-/γ-tocopherol, and retinol levels. Significant correlations with age were observed for lycopene (r = −0.248), α-tocopherol (r = 0.208), α-carotene (r = −0.112), and β-cryptoxanthin (r = 0.125; all p < 0.001). Age was inversely associated with lycopene (−6.5% per five-year age increase) and this association remained in the multiple regression model with the significant predictors (covariables) being country, season, cholesterol, gender, smoking status, body mass index (BMI (kg/m2)), and dietary habits. The positive association of α-tocopherol with age remained when all covariates including cholesterol and use of vitamin supplements were included (1.7% vs. 2.4% per five-year age increase). The association of higher β-cryptoxanthin with higher age was no longer statistically significant after adjustment for fruit consumption, whereas the inverse association of α-carotene with age remained in the fully adjusted multivariable model (−4.8% vs. −3.8% per five-year age increase). We conclude from our study that age is an independent predictor of plasma lycopene, α-tocopherol, and α-carotene.
BackgroundWheat is one of the most common food allergens in early childhood. In contrast to other food allergies, wheat-specific IgE correlates badly with clinical symptoms and relevant components have been identified mostly for wheat-depended exercise-induced anaphylaxis. Moreover, a high percentage of patients present with immediate type symptoms but wheat-specific IgE cannot be detected with commercial available systems. ObjectiveWe addressed the question whether the IgE recognition pattern between wheat allergic (WA) and clinically tolerant (WT) children differs in order to identify individual proteins useful for component-resolved diagnostics. MethodsSera of 106 children with suspected wheat allergy, of whom 44 children had clinical relevant wheat allergy and 62 were tolerant upon oral food challenge, were analyzed for wheat-specific IgE using the ImmunoCap system as well as immunoblots against water and salt soluble, and water-insoluble protein fractions. 40 randomly selected sera were analyzed for specific IgE to 5-gliadin. ResultsSixty-three percent of the WT and 86% of the WA children were sensitized to wheat with >0.35 kU(A)/l in ImmunoCAP analysis. We could confirm the role of -, ss-, -, and -gliadins, and LMW glutenin subunits as major allergens and found also IgE binding to a broad spectrum of water- and salt-soluble protein bands. It is of great importance that wheat allergic and tolerant patients showed IgE binding to the same protein bands. WT and WA did not significantly differ in levels of 5-gliadin-specific IgE. Conclusions & Clinical RelevanceChildren with challenge proven clinical relevant food allergy and tolerant ones had a similar spectrum of IgE binding to the same protein bands. These findings imply that component-resolved diagnostics might not be helpful in the diagnostic work-up of wheat allergy.