[Show abstract][Hide abstract] ABSTRACT: Food allergenic reactions have emerged as a growing challenge to the food industry. Soybean is considered an important allergenic food, which ranks among the so-called “big 8”, causing 90% of all immunoglobulin E (IgE)-mediated allergenic reactions worldwide1. The prevalence of food allergies is rising dramatically, leading to a great demand for methods aimed to reduce allergenic reactions. The technological approach hitherto has been focused on thermal methods, which commonly retain the ability of soy to elicit an immune reaction. More recently, novel nonthermal food processing methods are appealing in the food industry. However, the effect of these methods on soybean allergy has scarcely been investigated up to now.
The objective of this research activity was to study the effect of direct and indirect nonthermal atmospheric plasma (NTAP), pulsed ultraviolet (PUV) light, and γ-irradiation (3, 5, 10, 25, 50, 100 kGy) on the molecular weight distribution (SDS-PAGE) and immunoreactivity of soy protein isolates. Sandwich enzyme-linked immunosorbent assay (ELISA) and western blot using newly developed epitope-specific anti ß-conglycinin antibodies and human sera from soy sensitive individuals, respectively, were performed to measure the residual immunoreactivity of the resulting samples. In addition, sample weight, temperature, hydrogen peroxide content and pH value were measured.
SDS-PAGE results revealed that all processing methods showed significantly reduced protein intensities bands corresponding to the major soybean allergens (ß-conglycinin, glycinin). Sandwich ELISA results confirmed the significant reduction (P < 0.05) in IgE-binding for all methods. With an exception of indirect NTAP a reduction of up to 100% depending on treatment duration and dose rate could be observed. This attenuation was also examined by western blotting using serum from patients clinically tested to suffer from soy allergy.
The results suggest that all three nonthermal technologies could potentially be used to effectively reduce IgE-binding to soy proteins.
1 FDA (2004). Food Allergen and Labelling and Consumer Protection (FALCP) Act of 2004.
[Show abstract][Hide abstract] ABSTRACT: Soy is one of the most important valuable crops and provides the largest source of vegetable protein to human diet. However, soy is one of the “Big 8”, causing 90% of all immunoglobulin E (IgE)-mediated food allergies worldwide1. As food allergies emerged considerably in the last years, science and industry are searching for thermal and nonthermal technologies to control soybean allergy. So far, the use of conventional technologies - alone or combined - was not sufficient in the reduction of soybean allergy without affecting the functional and organoleptic properties. The present study presents a feasible technology to reduce allergenic potential of soybean. This study investigates the effects of high pressure processing (HPP) prior to and during enzymatic hydrolysis using the enzyme preparation Flavourzyme on the degree of hydrolysis (DH), molecular weight distribution (SDS-PAGE) and immunoreactivity of soy protein isolate (SPI). Experiments were carried out under atmospheric pressure (0.1 MPa) and HPP (100-600 MPa) at 50°C for 15 min. Residual immunoreactivity was measured by sandwich enzyme-linked immunosorbent assay (ELISA) using newly developed antibodies. Multivariate statistical principal component analysis (PCA) was employed on descriptive sensory data and the physicochemical properties were also analyzed. HPP greatly enhanced the hydrolysis of soy proteins as evidenced by an increase in DH and a sharp degradation of major soybean allergens (ß-conglycinin, glycinin) compared to hydrolysis under atmospheric pressure. Immunoreactivity was almost completely inhibited (500 MPa), applying HPP during enzymatic hydrolysis. The hydrolysates are well-suited as low-allergen foods as their smell, taste and mouthfeeling were even better than that of native SPI. The emulsifying and water-binding capacity remained unchanged compared to native SPI, whereas protein solubility, oil-binding capacity (2.6 mL g-1) and foaming activity (1603.7%) were improved. This technology represents an innovative approach to produce low-allergen foods that combine low immunoreactivity with superior physicochemical and sensory properties.
1 FDA (2004). Food Allergen and Labeling and Consumer Protection (FALCP) Act of 2004.
[Show abstract][Hide abstract] ABSTRACT: Food allergy is a major health concern and the incidence is dramatically rising. Soybean is one of the most important valuable crops in the world due to its considerable amount of high quality proteins. However, soybean is listed among the eight priority allergens, causing 90% of all Immunoglobulin E (IgE)-mediated food allergies. Recently, the need to control soybean allergy by methods other than avoidance has spurred the development of new technologies, including thermal and non-thermal treatments. Literature about the combined effect of high pressure (HP) and enzymatic hydrolysis on immunoreactivity of soy protein isolate (SPI) is scarce.
To contribute to this, the effects of HP applied prior to or during enzymatic hydrolysis of SPI by Flavourzyme on the extent of hydrolysis, immunoreactivity, and sensory characteristics were studied. Experiments were carried out under atmospheric pressure (0.1MPa) and HP (100-600MPa) at 50°C and pH 8.0 for 15min. The degree of hydrolysis (DH) was measured by the o-phthaldialdehyde (OPA) method and SDS-PAGE analysis. Further, newly developed antibodies were used for testing potential effects on consumers. The sensory perception, particular bitterness was evaluated by a professional panel on the basis of a 10-cm line scale, ranging from 0 (not detectable) to 10 (intense).
The results revealed that pressurization promoted the hydrolysis of Flavourzyme as evidenced by an increase in the DH value from an initial DH of 2.1% to 9.6%, whereas the DH of the hydroylsate prepared under atmospheric pressure was 5.4%. SDS-PAGE results showed changes in the molecular weight profile as β-conglycinin, P34, and the acidic subunit of glycinin were hydrolyzed in a varying degree, while bitterness remained unchanged. The hydrolysates were ranked according to reactivity in the developed immunoassay, leading to interesting results.
HP as an innovative non-thermal technology might give a contribution to the development of hypoallergenic products with low bitterness.
[Show abstract][Hide abstract] ABSTRACT: The demand for health promoting food products rapidly increases. In particular plant proteins play an important role in a healthy and sustainable diet. But many protein sources such as wheat and soy are not tolerated by many people and are listed among the eight priority allergens, causing 90% of all Immunoglobulin E (IgE)-mediated food allergies. As food intolerances emerged considerably in the last years, science and industry is searching for thermal and non-thermal technologies to control soybean allergy other than avoidance. The present work presents a new feasible method to sufficiently reduce allergy of the protein products.
Since soybeans are one of the most important grains among the legume family and a major source of high quality proteins, the effects of high pressure (HP) on soybean isolates - as an example - were studied.
HP was applied prior to and during the enzymatic hydrolysis of the soy protein isolates using a proteolytic enzyme preparation (Flavourzyme). The extent of hydrolysis, immunoreactivity, and sensory characteristics of the soy hydrolysates were investigated. Experiments were carried out under atmospheric pressure (0.1MPa) and HP (100-600MPa) at 50°C and pH 8.0 for 15min. The degree of hydrolysis (DH) was measured by the o-phthaldialdehyde method and SDS-PAGE analysis. Further, newly developed antibodies were used for testing potential immunological effects on consumers.
The results revealed that pressurization promoted the hydrolysis provoked by Flavourzyme as an increase in the DH value of 2.1% to 8.1% and 9.6% could be observed applying a pressure of 600MPa prior to or 100MPa during enzymatic hydrolysis. Instead, under atmospheric pressure a DH of 5.4% was detected after enzymatic hydrolysis. SDS-PAGE results clearly showed that HP effectively support the degradation of the main soybean allergens (conglycinin, glycinin, P34) compared to single enzymatic hydrolysis, where only slight changes were visible. The resulting hydrolysates were ranked according to IgE reactivity in the developed immunoassay and showed great reduction in soybean conglycinin up to 100%.
HP as an innovative non-thermal technology in combination with enzymatic hydrolysis can give a great contribution to the development of healthy and low-allergenic (hypoallergenic) products.
[Show abstract][Hide abstract] ABSTRACT: Unlabelled:
Reduction of β-catenin (CTNNB1) destroying complex components, for example, adenomatous polyposis coli (APC), induces β-catenin signaling and subsequently triggers activation of genes involved in proliferation and tumorigenesis. Though diminished expression of APC has organ-specific and threshold-dependent influence on the development of liver tumors in mice, the molecular basis is poorly understood. Therefore, a detailed investigation was conducted to determine the underlying mechanism in the development of liver tumors under reduced APC levels. Mouse liver at different developmental stages was analyzed in terms of β-catenin target genes including Cyp2e1, Glul, and Ihh using real-time RT-PCR, reporter gene assays, and immunohistologic methods with consideration of liver zonation. Data from human livers with mutations in APC derived from patients with familial adenomatous polyposis (FAP) were also included. Hepatocyte senescence was investigated by determining p16(INK4a) expression level, presence of senescence-associated β-galactosidase activity, and assessing ploidy. A β-catenin activation of hepatocytes does not always result in β-catenin positive but unexpectedly also in mixed and β-catenin-negative tumors. In summary, a senescence-inducing program was found in hepatocytes with increased β-catenin levels and a positive selection of hepatocytes lacking p16(INK4a), by epigenetic silencing, drives the development of liver tumors in mice with reduced APC expression (Apc(580S) mice). The lack of p16(INK4a) was also detected in liver tumors of mice with triggers other than APC reduction.
Epigenetic silencing of p16(Ink4a) in selected liver cells bypassing senescence is a general principle for development of liver tumors with β-catenin involvement in mice independent of the initial stimulus.
No preview · Article · Sep 2014 · Molecular Cancer Research
[Show abstract][Hide abstract] ABSTRACT: Aims:
Neurodegeneration in Alzheimer's disease (AD) is characterized by pathological protein aggregates and inadequate activation of cell cycle regulating proteins. Recently, Smad proteins were identified to control the expression of AD relevant proteins such as APP, CDK4 and CDK inhibitors, both critical regulators of cell cycle activation. This might indicate a central role for Smads in AD pathology where they show a substantial deficiency and disturbed subcellular distribution in neurones. Still, the mechanisms driving relocation and decrease of neuronal Smad in AD are not well understood. However, Pin1, a peptidyl-prolyl-cis/trans-isomerase, which allows isomerization of tau protein, was recently identified also controlling the fate of Smads. Here we analyse a possible role of Pin1 for Smad disturbances in AD.
Multiple immunofluorescence labelling and confocal laser-scanning microscopy were performed to examine the localization of Smad and Pin1 in human control and AD hippocampi. Ectopic Pin1 expression in neuronal cell cultures combined with Western blot analysis and immunoprecipitation allowed studying Smad level and subcellular distribution. Luciferase reporter assays, electromobility shift, RNAi-technique and qRT-PCR revealed a potential transcriptional impact of Smad on Pin1 promoter.
We report on a colocalization of phosphorylated Smad in AD with Pin1. Pin1 does not only affect Smad phosphorylation and stability but also regulates subcellular localization of Smad2 and supports its binding to phosphorylated tau protein. Smads, in turn, exert a negative feed-back regulation on Pin1.
Our data suggest both Smad proteins and Pin1 to be elements of a vicious circle with potential pathogenetic significance in AD.
No preview · Article · Jun 2014 · Neuropathology and Applied Neurobiology
[Show abstract][Hide abstract] ABSTRACT: Humanized tumor mice (HTM) were generated by the co-transplantation of human hematopoietic stem cells and human breast cancer cells overexpressing HER2 into neonatal NOD-scid IL2Rγ(null) (NSG) mice. These mice are characterized by the development of a human immune system in combination with human breast cancer growth. Due to concurrent transplantation into newborn mice, transfer of MHC-mismatched tumor cells resulted in solid coexistence and immune cell activation (CD4(+) T cells, natural killer cells, and myeloid cells), but without evidence for rejection. Histological staining of the spleen of HTM revealed co-localization of human antigen-presenting cells together with human T and B cells allowing MHC-dependent interaction, and thereby the generation of T cell-dependent antibody production. Here, we investigated the capability of these mice to generate human tumor-specific antibodies and correlated immunoglobulin titers with tumor outgrowth. We found detectable IgM and also IgG amounts in the serum of HTM, which apparently controlled tumor development when IgG serum concentrations were above 10 µg/ml. Western blot analyses revealed that the tumor-specific antibodies generated in HTM did not recognize HER2/neu antigens, but different, possibly relevant antigens for breast cancer therapy. In conclusion, HTM offer a novel approach to generate complete human monoclonal antibodies that do not require further genetic manipulation (e. g., humanization) for a potential application in humans. In addition, efficacy and safety of the generated antibodies can be tested in the same mouse model under human-like conditions. This might be of particular interest for cancer subtypes with no currently available antibody therapy.
[Show abstract][Hide abstract] ABSTRACT: The aryl hydrocarbon receptor (AhR) is a ligand-activated transcription factor, which mediates toxic responses to environmental pollutants, such as 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) and related compounds. Besides its well known role in induction of xenobiotic metabolizing enzymes, for instance CYP1A1, the AhR is also involved in tumour promotion in rodents although the underlying mechanisms are still poorly understood. Additionally, the AhR is known to regulate cellular proliferation, which might result in either inhibition or stimulation of proliferation depending on the cell-type studied. Potential targets in hepatocarcinogenesis are liver oval (stem/progenitor) cells. In the present work we analysed the effect of TCDD on proliferation in oval cells derived from mouse liver. We show that TCDD inhibits proliferation in these cells. In line, the amount of G0/G1 cells increases in response to TCDD. We further show that the expression of cyclin D1 and cyclin A is decreased, while p27 is increased. As a result, the retinoblastoma protein is not phosphorylated thereby inducing G0/G1 arrest. Pharmacological inhibition of the AhR and knock-down of AhR expression by RNA interference decreased the inhibitory effect on cell cycle and protein expression, indicating that the AhR at least partially mediates cell cycle arrest.
No preview · Article · Sep 2013 · Toxicology Letters
[Show abstract][Hide abstract] ABSTRACT: Label-free methods streamline quantitative proteomics of tissues by alleviating the need for metabolic labeling of proteins with stable isotopes. Here we detail and implement solutions to common problems in label-free data processing geared toward tissue proteomics by one-dimensional gel electrophoresis followed by liquid chromatography tandem mass spectrometry (geLC MS/MS). Our quantification pipeline showed high levels of performance in terms of duplicate reproducibility, linear dynamic range, and number of proteins identified and quantified. When applied to the liver of an adenomatous polyposis coli (APC) knockout mouse, we demonstrated an 8-fold increase in the number of statistically significant changing proteins compared to alternative approaches, including many more previously unidentified hydrophobic proteins. Better proteome coverage and quantification accuracy revealed molecular details of the perturbed energy metabolism.
No preview · Article · Jun 2012 · Journal of Proteome Research
[Show abstract][Hide abstract] ABSTRACT: Alzheimer's disease (AD) is a neurodegenerative disorder characterized by deregulation of neuronal cell cycle and differentiation control eventually resulting in cell death. During brain development, neuronal differentiation is regulated by Smad proteins, which are elements of the canonical transforming growth factor β (TGF-β) signaling pathway, linking receptor activation to gene expression. In the normal adult brain, Smad proteins are constitutively phosphorylated and predominantly localized in neuronal nuclei. Under neurodegenerative conditions such as AD, the subcellular localization of their phosphorylated forms is heavily disturbed, raising the question of whether a nuclear Smad deficiency in neurons might contribute to a loss of neuronal differentiation control and subsequent cell cycle re-entry. Here, we show by luciferase reporter assays, electromobility shift, and RNA interference (RNAi) technique a direct binding of Smad proteins to the CDK4 promoter inducing transcriptional inhibition of cell cycle-dependent kinase 4 (Cdk4). Mimicking the neuronal deficiency of Smad proteins observed in AD in cell culture by RNAi results in elevation of Cdk4 and retardation of neurite outgrowth. The results identify Smad proteins as direct transcriptional regulators of Cdk4 and add further evidence to a Smad-dependent deregulation of Cdk4 in AD, giving rise to neuronal dedifferentiation and cell death.
No preview · Article · Mar 2012 · Neurobiology of aging
[Show abstract][Hide abstract] ABSTRACT: Proliferation of oval cells, the bipotent precursor cells of the liver, requires impeded proliferation and loss of hepatocytes as well as a specific micro-environment, provided by adjacent sinusoidal cells of liver. Despite their immense importance for triggering the oval cell response, cells of hepatic sinusoids are rarely investigated. To elucidate the response of sinusoidal liver cells we have employed a choline-deficient, ethionine-supplemented (CDE) diet, a common method for inducing an oval cell response in rodent liver. We have utilised selected expression markers commonly used in the past for phenotypic discrimination of oval cells and sinusoidal cells: cytokeratin, E-cadherin and M2-pyruvate kinase for oval cells; and glial fibrillary acidic protein (GFAP) was used for hepatic stellate cells (HSCs).
CDE diet leads to an activation of all cells of the hepatic sinusoid in the mouse liver. Beside oval cells, also HSCs and Kupffer cells proliferate. The entire fraction of proliferating cells in mouse liver as well as endothelial cells and cholangiocytes express M2-pyruvate kinase. Concomitantly, GFAP, long considered a unique marker of quiescent HSCs was upregulated in activated HSCs and expressed also in cholangiocytes and oval cells.
Our results point to an important role of all types of sinusoidal cells in regeneration from CDE induced liver damage and call for utmost caution in using traditional marker for identifying specific cell types. Thus, M2-pyruvate kinase should no longer be used for estimating the oval cell response in mouse liver. CDE diet leads to activation of GFAP positive HSCs in the pericentral zone of liver lobulus. In the periportal zone the detection of GFAP in biliary cells and oval cells, calls other cell types as progenitors of hepatocytes into question under CDE diet conditions.
Full-text · Article · Oct 2010 · Comparative Hepatology
[Show abstract][Hide abstract] ABSTRACT: Adverse alcohol effects in the liver involve oxidative metabolism, fat deposition and release of fibrogenic mediators, including TGF-beta. The work presents an assessment of liver damaging cross-talk between ethanol and TGF-beta in hepatocytes.
To investigate TGF-beta effects on hepatocytes, microarray analyses were performed and validated by qRT-PCR, Western blot analysis and immunohistochemistry. The cellular state was determined by assessing lactate dehydrogenase, cellular glutathione, reactive oxygen species, lipid peroxidation and neutral lipid deposition. RNA interference was used for gene silencing in vitro.
TGF-beta is induced in mouse livers after chronic ethanol insult, enhances ethanol induced oxidative stress and toxicity towards cultured hepatocytes plus induces lipid-, oxidative stress metabolism- and fibrogenesis-gene expression signatures. Interestingly, TGF-beta down-regulates alcohol metabolizing enzyme Adh1 mRNA in cultured hepatocytes and liver tissue from TGF-beta transgenic mice via the ALK5/Smad2/3 signalling branch, with Smad7 as a potent negative regulator. ADH1 deficiency is a determining factor for the increased lipid accumulation and Cyp2E1 dependent toxicity in liver cells upon alcohol challenge. Further, ADH1 expression was decreased during liver damage in an intragastric ethanol infusion mouse model.
In the presence of ethanol, TGF-beta displays pro-steatotic action in hepatocytes via decreasing ADH1 expression. Low ADH1 levels are correlated with enhanced hepatocyte damage upon chronic alcohol consumption by favoring secondary metabolic pathways.
Full-text · Article · Mar 2010 · Journal of Hepatology
[Show abstract][Hide abstract] ABSTRACT: Smad2 and Smad3 are central molecules of the TGFbeta and activin receptor complex mediated intracellular signaling pathway. They function as important transcription factors playing essential roles in brain development. Interestingly they are also known to be involved in the pathogenesis of various neurological disorders (including Alzheimer's disease). Due to structural differences in the N-terminal Mad homology domain 1, Smad2 and Smad3 differ in their ability to bind DNA directly. A splice form of Smad2 lacking exon3, Smad2(Deltaexon3), assumes features of Smad3, in that it can directly bind to DNA resulting in a functional hybrid of Smad2 and Smad3 properties. There is very little information available on the expression of Smad2 isoforms in the brain. We report here that Smad2(Deltaexon3) is the most abundant of the two Smad2 isoforms in mouse brain and that Smad expression pattern alters during development and aging. Neuronal expression of Smad2(Deltaexon3) was confirmed by a single-cell PCR approach. Moreover, Smad2(Deltaexon3) predominates in the nuclear fraction of neurons, suggesting special function during brain differentiation. Our data indicate that there may be a specific role for Smad2(Deltaexon3) in neurons.
No preview · Article · May 2009 · International journal of developmental neuroscience: the official journal of the International Society for Developmental Neuroscience
[Show abstract][Hide abstract] ABSTRACT: The profibrogenic role of transforming growth factor (TGF)-beta in liver has mostly been attributed to hepatic stellate cell activation and excess matrix synthesis. Hepatocytes are believed to contribute to increased rates of apoptosis.
Primary hepatocyte outgrowths and AML12 cells were used as an in vitro model to detect TGF-beta effects on the cellular phenotype and expression profile. Furthermore, a transgenic mouse model was used to determine the outcome of hepatocyte-specific Smad7 expression on fibrogenesis following CCl(4)-dependent damage. Samples from patients with chronic liver diseases were assessed for (partial) epithelial-to-mesenchymal transition (EMT) in hepatocytes.
In primary cell cultures and in vivo, the majority of hepatocytes survive despite activated TGF-beta signaling. These cells display phenotypic changes and express proteins characteristic for (partial) EMT and fibrogenesis. Experimental expression of Smad7 in hepatocytes of mice attenuated TGF-beta signaling and EMT, resulted in less accumulation of interstitial collagens, and improved CCl(4)-provoked liver damage and fibrosis scores compared with controls.
The data indicate that hepatocytes undergo TGF-beta-dependent EMT-like phenotypic changes and actively participate in fibrogenesis. Furthermore, ablation of TGF-beta signaling specifically in this cell type is sufficient to blunt the fibrogenic response.
[Show abstract][Hide abstract] ABSTRACT: Terminal differentiation requires molecules also involved in aging such as the cell cycle inhibitor p16(INK4a). Like other organs, the adult liver represents a quiescent organ with terminal differentiated cells, hepatocytes and cholangiocytes. These cells retain the ability to proliferate in response to liver injury or reduction of liver mass. However, under conditions which prevent mitotic activation of hepatocytes, regeneration can occur instead from facultative hepatic stem cells.For therapeutic application a non-toxic activation of this stem cell compartment is required. We have established transgenic mice with conditional overexpression of the cell cycle inhibitor p16(INK4a) in hepatocytes and have provoked and examined oval cell activation in adult liver in response to a range of proliferative stimuli. We could show that the liver specific expression of p16(INK4a) leads to a faster differentiation of hepatocytes and an activation of oval cells already in postnatal mice without negative consequences on liver function.
No preview · Article · Apr 2008 · Journal of Cellular and Molecular Medicine