Wiebke Schlörmann

The shape of caveolae is omega-like after glutaraldehyde fixation and cup-like after cryofixation

Caveolae were defined as flask- or omega-shaped plasma membrane invaginations, abundant in adipocytes, fibroblasts, endothelial and smooth muscle cells. The major protein component of caveolar membranes is an integral membrane protein named caveolin. We compared the freeze-fracture behavior of caveolae in glutaraldehyde-fixed and cryofixed mouse fibroblast cells and found distinct differences. In glutaraldehyde-fixed cells almost all caveolae were cross-fractured through their pore and only very few caveolar membranes were membrane-fractured. We found the reverse situation in rapid frozen cells without any chemical fixation where most of the caveolae were membrane-fractured, showing different degrees of invagination from nearly flat to deeply invaginated. In ultrathin sections of glutaraldehyde-fixed heart endothelial cells, caveolae exhibit the well known omega-like shape. In high-pressure frozen, freeze-substituted and low temperature embedded heart endothelial cells, the caveolae frequently exhibit a cup-like shape without any constriction or pore. The cup-like caveolar shape could also be shown by tilt series analysis of freeze-fracture replicas obtained from cryofixed cells. Freeze-fracture immunolabeling of caveolin-1 revealed a lateral belt-like caveolin alignment. These findings point out that the constricted “neck” region of caveolae in most cases is an effect that is caused and intensified by the glutaraldehyde fixation. Our data indicate that caveolae in vivo show all degrees of invagination from nearly flat via cup-like depressed to in a few cases omega-like.

Influence of roasting conditions on health-related compounds in different nuts.

Due to their health-beneficial ingredients the consumption of nuts can contribute to a healthy diet. The composition of hazelnuts, almonds, macadamia nuts, pistachios and walnuts regarding health-promoting and potentially harmful compounds was examined before and after roasting under different time and temperature conditions. Fatty acid compositions were not affected by roasting. Malondialdehyde increased with higher roasting temperatures (17-fold in walnuts). Levels of tocopherol isomers were reduced after roasting (α-T: 38%, β-T: 40%, γ-T: 70%) and hydrophilic antioxidant capacity decreased significantly in hazelnuts (1.4-fold), macadamia nuts (1.7-fold) and walnuts (3.7-fold). Increasing roasting temperatures supported the formation of significant amounts of acrylamide only in almonds (1220 μg kg(-1)). In general, nuts roasted at low/middle temperatures (120-160°C) exhibited best sensory properties. Therefore, desired sensory quality along with a favourable healthy nut composition may be achieved by roasting over a low to medium temperature range.

Vaccenic acid-mediated reduction in cytokine production is independent of c9,t11-CLA in human peripheral blood mononuclear cells.

The ruminant trans fatty acid vaccenic acid (tVA) favorably alters markers of inflammation. However, it is not yet clear whether these effects are attributed to its endogenous partial conversion to c9,t11-CLA, which is known to possess anti-inflammatory properties. We compared the cytokine reducing potential of tVA to c9,t11-CLA in human T-helper (Th) cells as a main source of cytokine production during inflammation. Secondly, we assessed whether a bioconversion of tVA to c9,t11-CLA via stearoyl-CoA desaturase (SCD) encoded activity takes place in peripheral blood mononuclear cells (PBMC) in order to relate the outcomes of intracellular cytokine measurement to the degree of conversion. TVA reduced the percentage of both IL-2 and TNF-α expressing Th cells significantly, but to a lesser extent compared to c9,t11-CLA, as determined by flow cytometry after alloreactive stimulation of PBMC. Pre-treatment with the selective PPARγ antagonist T0070907 largely re-established the IL-2 and TNF-α positive Th cell population in both tVA and c9,t11-CLA treated cultures. Interestingly, while the portion of tVA dose-dependently increased within the cellular lipid fraction, the initially marginal amount of c9,t11-CLA remained unaltered. However, SCD mRNA although abundantly expressed in PBMC was not regulated by tVA. Conclusively, these results suggest that the cytokine reducing effect of tVA in human T cells is independent of c9,t11-CLA, since no bioconversion occurred. Moreover, the data provide evidence that tVA mechanistically acts in a manner similar to c9,t11-CLA.

Chemopreventive effects of in vitro digested and fermented bread in human colon cells

PurposeBread as a staple food product represents an important source for dietary fibre consumption. Effects of wheat bread, wholemeal wheat bread and wholemeal rye bread on mechanisms which could have impact on chemoprevention were analysed in colon cells after in vitro fermentation.MethodsEffects of fermented bread samples on gene expression, glutathione S-transferase activity and glutathione content, differentiation, growth and apoptosis were investigated using the human colon adenoma cell line LT97. Additionally, apoptosis was studied in normal and tumour colon tissue by determination of caspase activities.ResultsThe expression of 76 genes (biotransformation, differentiation, apoptosis) was significantly upregulated (1.5-fold) in LT97 cells. The fermented bread samples were able to significantly increase glutathione S-transferase activity (1.8-fold) and glutathione content (1.4-fold) of the cells. Alkaline phosphatase activity as a marker of differentiation was also significantly enhanced (1.7-fold). The fermented bread samples significantly inhibited LT97 cell growth and increased the level of apoptotic cells (1.8-fold). Only marginal effects on apoptosis in tumour compared to normal tissue were observed.ConclusionsThis is the first study which presents chemopreventive effects of different breads after in vitro fermentation. In spite of differences in composition, the results were comparable between the bread types. Nevertheless, they indicate a potential involvement of this staple food product regarding the prevention of colon cancer.

B-vitamins, carotenoids and α-/γ-tocopherol in raw and roasted nuts

The concentrations of B-vitamins, carotenoids and tocopherols in nuts may differ between species and might be influenced by roasting. Thiamine, riboflavin, pyridoxine, lutein, zeaxanthin, β-carotene and α-/γ-tocopherol were determined in different varieties of raw and roasted nuts using HPLC (fluorescence/UV-vis detection). The analysis revealed remarkable concentrations of thiamine and pyridoxine in pistachios (57%, 79% of the recommended daily intake/100g (RDI), respectively) and riboflavin in almonds (119% of the RDI). Pistachios were rich in lutein/zeaxanthin and contained highest β-carotene levels among nuts. Almonds and hazelnuts were abundant in α-tocopherol (>4-fold the RDI for tocopherol equivalents) while pistachios and walnuts were rich in γ-tocopherol. Roasting had a diminishing effect on thiamine, carotenoids and tocopherols especially in almonds and walnuts. Nuts could make a valuable contribution to a healthy diet in regard to B-vitamins, lutein/zeaxanthin and tocopherols. A reduction in micronutrient content by roasting is reliant on the nut variety and specific micronutrient.

In vitro fermented nuts exhibit chemopreventive effects in HT29 colon cancer cells

It is proven that nuts contain essential macro- and micronutrients, e.g. fatty acids, vitamins and dietary fibre (DF). Fermentation of DF by the gut microflora results in the formation of SCFA which are recognised for their chemopreventive potential, especially by influencing cell growth. However, little is known about cellular response to complex fermentation samples of nuts. Therefore, we prepared and analysed (pH, SCFA, bile acids, tocopherol, antioxidant capacity) fermentation supernatant (fs) fractions of nuts (almonds, macadamias, hazelnuts, pistachios, walnuts) after in vitro fermentation and determined their effects on growth of HT29 cells as well as their genotoxic/anti-genotoxic potential. The fermented nut samples contained 2- to 3-fold higher amounts of SCFA than the faeces control, but considerable reduced levels of bile acids. While most of the investigated native nuts comprised relatively high amounts of tocopherol (α-tocopherol in almonds and hazelnuts and γ- and δ-tocopherol in pistachios and walnuts), rather low concentrations were found in the fs. All nut extracts and nut fs showed a strong antioxidant potential. Furthermore, all fs, except the fs pistachio, reduced growth of HT29 cells significantly. DNA damage induced by H₂O₂ was significantly reduced by the fs of walnuts after 15 min co-incubation of HT29 cells. In conclusion, this is the first study which presents the chemopreventive effects (reduction of tumour-promoting desoxycholic acid, rise in chemopreventive SCFA, protection against oxidative stress) of different nuts after in vitro digestion and fermentation, and shows the potential importance of nuts in the prevention of colon cancer.

Foetal cord blood contains higher portions of n-3 and n-6 long-chain PUFA but lower portions of trans C18:1 isomers than maternal blood

Background/objective An adequate supply of long-chain polyunsaturated fatty acids (LC PUFA) promotes foetal health and development, whereas generally, trans fatty acids (tFA) are considered to negatively interfere with LC PUFA metabolism. Nevertheless, to date, limited data concerning separate trans C18:1, such as t9 and t11, are available for maternal and foetal blood. Therefore, in this study the portions of individual trans C18:1, LC n-6, and n-3 PUFA in lipids of maternal and foetal plasma and erythrocyte membranes of German mother and child pairs (n=40) were analysed. Results Portions of linoleic acid and α-linolenic acid as LC precursors were lower (~0.4-fold); whereas the metabolites arachidonic acid (AA, n-6) and docosahexaenoic acid (DHA, n-3) were significantly higher (~2-fold) in foetal than in maternal plasma and erythrocytes. The main tFA in maternal and foetal blood were elaidic acid (C18:1t9; t9) and vaccenic acid (C18:1t11; t11). Portions of t9, t10, t11, and t12 in foetal blood lipids were lower (~0.5-fold) compared with maternal blood. In foetal lipids, t9 was higher than t11. The t9 correlated negatively with eicosapentaenoic acid (n-3) and AA in maternal and foetal lipids; whereas t11 correlated negatively only with foetal total LC n-6 (plasma and erythrocytes) and n-3 PUFA (erythrocytes). No correlation between maternal tFA and foetal PUFA was observed. Conclusions ‘Biomagnification’ of LC n-6 and n-3 PUFA AA and DHA in foetal blood was confirmed, whereas single trans isomers were lower compared with maternal blood. Nevertheless, tFA intake, especially from industrial sources, should be as low as possible.

Comet fluorescence in situ hybridization (Comet-FISH): detection of DNA damage.

The Comet-FISH technique is a useful tool for detection of overall and region-specific DNA damage and repair in individual cells. It combines two well-established methods, the Comet assay (single-cell gel electrophoresis) and fluorescence in situ hybridization (FISH). The Comet assay serves as the basis of Comet-FISH and is a relatively simple and fast method that allows separation of fragmented from nonfragmented DNA and quantification of DNA damage and repair. FISH enables detection of specifically labeled DNA sequences of interest, including whole chromosomes. The combined technique of Comet-FISH is a modification of the Comet assay that inserts a hybridization step after unwinding and electrophoresis and permits the labeling of specific gene sequences or telomeres. Comet-FISH has been applied for detection of site-specific breaks in DNA regions that are relevant for development of various diseases, and has also been used to study the distribution of DNA damage and repair in the complete genome. Moreover, DNA sequence modifications can be detected in individual cells using Comet-FISH. Whereas results from the Comet assay alone reflect only the level of overall DNA damage, the addition of the FISH technique allows the assignment of the probed sequences to the damaged or undamaged part of the comet (tail or head, respectively).

Detection of DNA Damage by Comet Fluorescence In Situ Hybridization

Comet fluorescence in situ hybridization (Comet-FISH) is a useful method to detect overall and region-specific DNA damage in individual cells. Two well-established methods are combined, the Comet assay (single cell gel electrophoresis) and fluorescence in situ hybridization (FISH). The Comet assay is the method of choice for the detection of DNA damage. With the alkaline version the influence of specific substances such as water pollutants or ingredients of food on individual cells can be easily measured. The Comet assay involves the embedding of cells in agarose on microscopic slides, lysis of cells, and separation of DNA via electrophoresis. Damaged DNA migrates from the nucleus (head of the comet) forming a tail. The percentage of DNA in the tail correlates with the degree of DNA strand breaks (DNA damage). The combination of FISH with the Comet assay uses labeled probes which hybridize specifically to selected DNA sequences. This allows the detection of specific DNA damage or repair capacity in single cells. Here we present exemplarily the Comet-FISH method by detection of DNA damage using hydrogen peroxide as a genotoxic model substrate.

Chemopreventive potential of in vitro fermented nuts in LT97 colon adenoma and primary epithelial colon cells

Due to their beneficial nutritional profile the consumption of nuts contributes to a healthy diet and might reduce colon cancer risk. To get closer insights into potential mechanisms, the chemopreventive potential of different in vitro fermented nut varieties regarding the modulation of genes involved in detoxification (CAT, SOD2, GSTP1, GPx1) and cell cycle (p21, cyclin D2) as well as proliferation and apoptosis was examined in LT97 colon adenoma and primary epithelial colon cells. Fermentation supernatants (FS) of nuts significantly induced mRNA expression of CAT (up to 4.0‐fold), SOD2 (up to 2.5‐fold), and GSTP1 (up to 2.3‐fold), while GPx1 expression was significantly reduced by all nut FS (0.8 fold on average). Levels of p21 mRNA were significantly enhanced (up to 2.6‐fold), whereas all nut FS significantly decreased cyclin D2 expression (0.4‐fold on average). In primary epithelial cells, expression of CAT (up to 3.5‐fold), GSTP1 (up to 3.0‐fold), and GPx1 (up to 3.9‐fold) was increased, whereas p21 and cyclin D2 levels were not influenced. Nut FS significantly inhibited growth of LT97 cells and increased levels of early apoptotic cells (8.4% on average) and caspase 3 activity (4.6‐fold on average), whereas caspase 3 activity was not modulated in primary colon cells. The differential modulation of genes involved in detoxification and cell cycle together with an inhibition of proliferation and induction of apoptosis in adenoma cells might contribute to chemopreventive effects of nuts regarding colon cancer.