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Sphingolipids are major components of the plasma membrane. In particular, ceramide serves as an essential building hub for complex sphingolipids, but also as an organizer of membrane domains segregating receptors and signalosomes. Sphingomyelin breakdown as a result of sphingomyelinase activation after ligation of a variety of receptors is the predominant source of ceramides released at the plasma membrane. This especially applies to T lymphocytes where formation of ceramide-enriched membrane microdomains modulates TCR signaling. Because ceramide release and redistribution occur very rapidly in response to receptor ligation, novel tools to further study these processes in living T cells are urgently needed. To meet this demand, we synthesized nontoxic, azido-functionalized ceramides allowing for bio-orthogonal click-reactions to fluorescently label incorporated ceramides, and thus investigate formation of ceramide-enriched domains. Azido-functionalized C-6-ceramides were incorporated into and localized within plasma membrane microdomains and proximal vesicles in T cells. They segregated into clusters after TCR, and especially CD28 ligation, indicating efficient sorting into plasma membrane domains associated with T cell activation; this was abolished upon sphingomyelinase inhibition. Importantly, T cell activation was not abrogated upon incorporation of the compound, which was efficiently excluded from the immune synapse center as has previously been seen in Ab-based studies using fixed cells. Therefore, the functionalized ceramides are novel, highly potent tools to study the subcellular redistribution of ceramides in the course of T cell activation. Moreover, they will certainly also be generally applicable to studies addressing rapid stimulation-mediated ceramide release in living cells.
Reward-dependent instrumental behavior must continuously be re-adjusted according to environmental conditions. Failure to adapt to changes in reward contingencies may incur psychiatric disorders like anxiety and depression. When an expected reward is omitted, behavior undergoes extinction. While extinction involves active re-learning, it is also accompanied by emotional behaviors indicative of frustration, anxiety, and despair (extinction-induced depression). Here, we report evidence for a sphingolipid mechanism in the extinction of behavior. Rapid extinction, indicating efficient re-learning, coincided with a decrease in the activity of the enzyme acid sphingomyelinase (ASM), which catalyzes turnover of sphingomyelin to ceramide, in the dorsal hippocampus of rats. The stronger the decline in ASM activity, the more rapid was the extinction. Sphingolipid-focused lipidomic analysis showed that this results in a decline of local ceramide species in the dorsal hippocampus. Ceramides shape the fluidity of lipid rafts in synaptic membranes and by that way can control neural plasticity. We also found that aging modifies activity of enzymes and ceramide levels in selective brain regions. Aging also changed how the chronic treatment with corticosterone (stress) or intranasal dopamine modified regional enzyme activity and ceramide levels, coinciding with rate of extinction. These data provide first evidence for a functional ASM-ceramide pathway in the brain involved in the extinction of learned behavior. This finding extends the known cellular mechanisms underlying behavioral plasticity to a new class of membrane-located molecules, the sphingolipids, and their regulatory enzymes, and may offer new treatment targets for extinction- and learning-related psychopathological conditions.
Malnutrition, poor health, hunger, and even starvation are still the world's greatest challenges. Malnutrition is defined as deficiency of nutrition due to not ingesting the proper amounts of nutrients by simply not eating enough food and/or by consuming nutrient-poor food in respect to the daily nutritional requirements. Moreover, malnutrition and disease are closely associated and incidences of such diet-related diseases increase particularly in low- and middle-income states. While foods of animal origin are often unaffordable to low-income families, various neglected crops can offer an alternative source of micronutrients, vitamins, as well as health-promoting secondary plant metabolites. Therefore, agricultural and horticultural research should develop strategies not only to produce more food, but also to improve access to more nutritious food. In this context, one promising approach is to promote biodiversity in the dietary pattern of low-income people by getting access to nutritional as well as affordable food and providing recommendations for food selection and preparation. Worldwide, a multitude of various plant species are assigned to be consumed as grains, vegetables, and fruits, but only a limited number of these species are used as commercial cash crops. Consequently, numerous neglected and underutilized species offer the potential to diversify not only the human diet, but also increase food production levels, and, thus, enable more sustainable and resilient agro- and horti-food systems. To exploit the potential of neglected plant (NP) species, coordinated approaches on the local, regional, and international level have to be integrated that consequently demand the involvement of numerous multi-stakeholders. Thus, the objective of the present review is to evaluate whether NP species are important as “Future Food” for improving the nutritional status of humans as well as increasing resilience of agro- and horti-food systems.
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.
The visceral protein transthyretin (TTR) is frequently affected by oxidative post-translational protein modifications (PTPMs) in various diseases. Thus, better insight into structure-function relationships due to oxidative PTPMs of TTR should contribute to the understanding of pathophysiologic mechanisms. While the in vivo analysis of TTR in mammalian models is complex, time- and resource-consuming, transgenic Caenorhabditis elegans expressing hTTR provide an optimal model for the in vivo identification and characterization of drug-mediated oxidative PTPMs of hTTR by means of matrix assisted laser desorption/ionization – time of flight – mass spectrometry (MALDI-TOF-MS). Herein, we demonstrated that hTTR is expressed in all developmental stages of Caenorhabditis elegans, enabling the analysis of hTTR metabolism during the whole life-cycle. The suitability of the applied model was verified by exposing worms to D-penicillamine and menadione. Both drugs induced substantial changes in the oxidative PTPM pattern of hTTR. Additionally, for the first time a covalent binding of both drugs with hTTR was identified and verified by molecular modelling.
The visceral protein transthyretin (TTR) is frequently affected by oxidative post-translational protein modifications (PTPMs) in various diseases. Thus, better insight into structure-function relationships due to oxidative PTPMs of TTR should contribute to the understanding of pathophysiologic mechanisms. While the in vivo analysis of TTR in mammalian models is complex, time- and resource-consuming, transgenic Caenorhabditis elegans expressing hTTR provide an optimal model for the in vivo identification and characterization of drug-mediated oxidative PTPMs of hTTR by means of matrix assisted laser desorption/ionization – time of flight – mass spectrometry (MALDI-TOF-MS). Herein, we demonstrated that hTTR is expressed in all developmental stages of Caenorhabditis elegans, enabling the analysis of hTTR metabolism during the whole life-cycle. The suitability of the applied model was verified by exposing worms to D-penicillamine and menadione. Both drugs induced substantial changes in the oxidative PTPM pattern of hTTR. Additionally, for the first time a covalent binding of both drugs with hTTR was identified and verified by molecular modelling.
The visceral protein transthyretin (TTR) is frequently affected by oxidative post-translational protein modifications (PTPMs) in various diseases. Thus, better insight into structure-function relationships due to oxidative PTPMs of TTR should contribute to the understanding of pathophysiologic mechanisms. While the in vivo analysis of TTR in mammalian models is complex, time-and resource-consuming, transgenic Caenorhabditis elegans expressing hTTR provide an optimal model for the in vivo identification and characterization of drug-mediated oxidative PTPMs of hTTR by means of matrix assisted laser desorption/ionization - time of flight - mass spectrometry (MALDI-TOF-MS). Herein, we demonstrated that hTTR is expressed in all developmental stages of Caenorhabditis elegans, enabling the analysis of hTTR metabolism during the whole life-cycle. The suitability of the applied model was verified by exposing worms to D-penicillamine and menadione. Both drugs induced substantial changes in the oxidative PTPM pattern of hTTR. Additionally, for the first time a covalent binding of both drugs with hTTR was identified and verified by molecular modelling.
Exosomes are small membrane vesicles released by different cell types, including hepatocytes, that play important roles in intercellular communication. We have previously demonstrated that hepatocyte-derived exosomes contain the synthetic machinery to form sphingosine-1-phosphate (S1P) in target hepatocytes resulting in proliferation and liver regeneration after ischemia/reperfusion (I/R) injury. We also demonstrated that the chemokine receptors, CXCR1 and CXCR2, regulate liver recovery and regeneration after I/R injury. In the current study, we sought to determine if the regulatory effects of CXCR1 and CXCR2 on liver recovery and regeneration might occur via altered release of hepatocyte exosomes. We found that hepatocyte release of exosomes was dependent upon CXCR1 and CXCR2. CXCR1-deficient hepatocytes produced fewer exosomes, whereas CXCR2-deficient hepatocytes produced more exosomes compared to their wild-type controls. In CXCR2-deficient hepatocytes, there was increased activity of neutral sphingomyelinase (Nsm) and intracellular ceramide. CXCR1-deficient hepatocytes had no alterations in Nsm activity or ceramide production. Interestingly, exosomes from CXCR1-deficient hepatocytes had no effect on hepatocyte proliferation, due to a lack of neutral ceramidase and sphingosine kinase. The data demonstrate that CXCR1 and CXCR2 regulate hepatocyte exosome release. The mechanism utilized by CXCR1 remains elusive, but CXCR2 appears to modulate Nsm activity and resultant production of ceramide to control exosome release. CXCR1 is required for packaging of enzymes into exosomes that mediate their hepatocyte proliferative effect.
In this study, the applicability of semi-direct cold atmospheric pressure plasma (CAPP) during postharvest processing of Tenebrio molitor flour is investigated. Besides analyzing the decontamination efficacy, plasma induced impact on techno-functionality, protein solubility, composition and structure was determined and compared to heat induced effects. Following CAPP treatment, the total microbial load of the Tenebrio flour of 7.72 log(10) cfu/g was reduced to 7.10 (1 min), 6.72 (2.5 min), 5.79 (5 min), 5.19 (7.5 min), 521 (10 min) and 4.73 (15 min) log(10) cfu/g. With increasing exposure to CAPP, protein solubility at pH 4 almost linearly decreased to a minimum of 54%. Water binding capacity decreased from 0.79 to 0.64 gwatedg whereas oil binding capacity increased from 0.59 to 0.66 g(oil)/g. Gel electrophoresis revealed a decrease of all protein fractions at pH 4 whereas at pH 10 the band pattern significantly shifted to protein fractions with higher molecular weights. Industrial relevance: Edible insects are rich in valuable protein, fat, fibre, minerals and micronutrients. Although a wide range of species represent a valuable alternative protein source that could contribute to food and feed security, they are industrially hardly exploited. The tailored application of proper processing technologies could lead to novel insect-based high-protein food and feed products with unique functional properties supporting the increase in acceptability among potential consumers. Current research concentrates on developing processing chains including innovative nonthermal approaches. Cold atmospheric pressure plasma (CAPP) has gained attention as an effective technology for the decontamination and modification of fresh and dry agricultural products. In the postharvest chain of edible insects, the application of CAPP could contribute to the development of safe and high-quality insect-based products in the food and feed sector. (C) 2016 Published by Elsevier Ltd.
The concept of developmental origins of diseases has gained a huge interest in recent years and is a constantly emerging scientific field. First observations hereof originated from epidemiological studies, linking impaired birth outcomes to adult chronic, noncommunicable disease. By now there is a considerable amount of both epidemiological and experimental evidence highlighting the impact of early life events on later life disease susceptibility. Albeit far from being completely understood, more recent studies managed to elucidate underlying mechanisms, with epigenetics having become almost synonymous with developmental programming. The aim of this review was to give a comprehensive overview of various aspects and mechanisms of developmental origins of diseases. Starting from initial research foci mainly centered on a nutritionally impaired intrauterine environment, more recent findings such as postnatal nutrition, preterm birth, paternal programming and putative interventional approaches are summarized. The review outlines general underlying mechanisms and particularly discusses mechanistic explanations for sexual dimorphism in developmental programming. Furthermore, novel hypotheses are presented emphasizing a non-mendelian impact of parental genes on the offspring's phenotype.
Scope: The trace element selenium (Se) is an integral component of our diet. However, its metabolism and toxicity following elevated uptake are not fully understood. Since the either adverse or beneficial health effects strongly depend on the ingested Se species, five low molecular weight species were investigated regarding their toxicological effects, cellular bioavailability and species-specific metabolism in human cells. Methods and results: For the first time, the urinary metabolites methyl-2-acetamido-2-deoxy1- seleno-beta-D-galactopyranoside (selenosugar 1) and trimethylselenonium ion (TMSe) were toxicologically characterised in comparison to the food relevant species methylselenocysteine (MeSeCys), selenomethionine (SeMet) and selenite in human urothelial, astrocytoma and hepatoma cells. In all cell lines selenosugar 1 and TMSe showed no cytotoxicity. Selenite, MeSeCys and SeMet exerted substantial cytotoxicity, which was strongest in the urothelial cells. There was no correlation between the potencies of the respective toxic effects and the measured cellular Se concentrations. Se speciation indicated that metabolism of the respective species is likely to affect cellular toxicity. Conclusion: Despite being taken up, selenosugar 1 and TMSe are non-cytotoxic to urothelial cells, most likely because they are not metabolically activated. The absent cytotoxicity of selenosugar 1 and TMSe up to supra-physiological concentrations, support their importance as metabolites for Se detoxification.
Lutein and its isomer zeaxanthin have gained considerable interest as possible nutritional ingredient in the prevention of age-related macular degeneration (AMD) in humans. Egg yolk is a rich source of these carotenoids. As an oxidative sensitive component, antioxidants such as -tocopherol (T) might contribute to an improved accumulation in egg yolk. To test this, chickens were fed lutein esters (LE) with and without -tocopherol as an antioxidant. After depletion on a wheat-soya bean-based lutein-poor diet for 21days, laying hens (n=42) were equally divided into three groups and fed the following diets for 21days: control (basal diet), a LE group (40mg LE/kg feed) and LE+T group (40mg LE plus 100mg T/kg feed). Eggs and blood were collected periodically. Carotenoids and -tocopherol in yolk and blood plasma were determined by HPLC. Egg yolk was also analysed for total carotenoids using a one-step spectrophotometric method (iCheck(())). Lutein, zeaxanthin, -tocopherol and total carotenoids in egg yolk were highest after 14days of feeding and decreased slightly afterwards. At the end of the trial, eggs of LE+T group contained higher amount of lutein (13.72), zeaxanthin (0.65), -tocopherol (297.40) and total carotenoids (21.6) compared to the LE group (10.96, 0.55, 205.20 and 18.0mg/kg, respectively, p<0.05). Blood plasma values of LE+T group contain higher lutein (1.3), zeaxanthin (0.06) and tocopherol (20.1) compared to LE group (1.02, 0.04 and 14.90mg/l, respectively, p<0.05). In conclusion, dietary -tocopherol enhances bioavailability of lutein reflecting higher content in egg yolk and blood plasma. Improved bioavailability might be due to increased absorption of lutein in the presence of tocopherol and/or a greater stability of lutein/zeaxanthin due to the presence of -tocopherol as an antioxidant.
Despite high-dose vitamin A supplementation of very low birth weight infants (VLBW, <1500 g), their vitamin A status does not improve substantially. Unknown is the impact of urinary retinol excretion on the serum retinol concentration in these infants. Therefore, the effect of high-dose vitamin A supplementation on the urinary vitamin A excretion in VLBW infants was investigated. Sixty-three VLBW infants were treated with vitamin A (5000 IU intramuscular, 3 times/week for 4 weeks); 38 untreated infants were classified as control group. On days 3 and 28 of life, retinol, retinol-binding protein 4 (RBP4), glomerular filtration rate, proteinuria, and Tamm-Horsfall protein were quantified in urine. On day 3 of life, substantial retinol and RBP4 losses were found in both groups, which significantly decreased until day 28. Notwithstanding, the retinol excretion was higher (P<0.01) under vitamin A supplementation as compared to infants of the control group. On day 28 of life, the urinary retinol concentrations were predictive for serum retinol concentrations in the vitamin A treated (P<0.01), but not in the control group (P=0.570). Conclusion: High urinary retinol excretion may limit the vitamin A supplementation efficacy in VLBW infants. Advanced age and thus postnatal kidney maturation seems to be an important contributor in the prevention of urinary retinol losses.
Background: Transport of methylmercury (MeHg) across the blood-brain barrier towards the brain side is well discussed in literature, while ethylmercury (EtHg) and inorganic mercury are not adequately characterized regarding their entry into the brain. Studies investigating a possible efflux out of the brain are not described to our knowledge. Methods: This study compares, for the first time, effects of organic methylmercury chloride (MeHgCl), EtHg-containing thiomersal and inorganic Hg chloride (HgCl2) on as well as their transfer across a primary porcine in vitro model of the blood-brain barrier. Results: With respect to the barrier integrity, the barrier model exhibited a much higher sensitivity towards HgCl2 following basolateral incubation (brain-facing side) as compared to apical application (blood-facing side). These HgCl2 induced effects on the barrier integrity after brain side incubation are comparable to that of the organic species, although MeHgCl and thiomersal exerted much higher cytotoxic effects in the barrier building cells. Hg transfer rates following exposure to organic species in both directions argue for diffusion as transfer mechanism. Inorganic Hg application surprisingly resulted in a Hg transfer out of the brain-facing compartment. Conclusions: In case of MeHgCl and thiomersal incubation, mercury crossed the barrier in both directions, with a slight accumulation in the basolateral, brain-facing compartment, after simultaneous incubation in both compartments. For HgCl2, our data provide first evidence that the blood-brain barrier transfers mercury out of the brain.
Understanding penetration not only in intact, but also in lesional skin with impaired skin barrier function is important, in order to explore the surplus value of nanoparticle-based drug delivery for anti-inflammatory dermatotherapy. Herein, short-termex vivo cultures of (i) intact human skin, (ii) skin pretreated with tape-strippings and (iii) skin pre-exposed to sodium lauryl sulfate (SLS) were used to assess the penetration of dexamethasone (Dex). Intradermal microdialysis was utilized for up to 24 h after drug application as commercial cream, nanocrystals or ethyl cellulose nanocarriers applied at the therapeutic concentration of 0.05%, respectively. In addition, Dex was assessed in culture media and extracts from stratum corneum, epidermis and dermis after 24 h, and the results were compared to those in heat-separated split skin from studies in Franz diffusion cells. Providing fast drug release, nanocrystals significantly accelerated the penetration of Dex. In contrast to the application of cream and ethyl cellulose nanocarriers, Dex was already detectable in eluates after 6 h when applying nanocrystals on intact skin. Disruption of the skin barrier further accelerated and enhanced the penetration. Encapsulation in ethyl cellulose nanocarriers delayed Dex penetration. Interestingly, for all formulations highly increased concentrations in the dialysate were observed in tape-stripped skin, whereas the extent of enhancement was less in SLS-exposed skin. The results were confirmed in tissue extracts and were in line with the predictions made by in vitro release studies and ex vivo Franz diffusion cell experiments. The use of 45 kDa probes further enabled the collection of inflammatory cytokines. However, the estimation of glucocorticoid efficacy by Interleukin (IL)-6 and IL-8 analysis was limited due to the trauma induced by the probe insertion. Ex vivo intradermal microdialysis combined with culture media analysis provides an effective, skin-sparing method for preclinical assessment of novel drug delivery systems at therapeutic doses in models of diseased skin. (C) 2016 Elsevier B.V. All rights reserved.