Browsing by Subject "Oligosaccharide"

Now showing 1 - 2 of 2

Einfluss kurzkettiger Fettsäuren und mikrobieller Fermentationsprodukte neuartiger Oligosaccharide auf Cytotoxizität, Proliferation und Apoptose von humanen Coloncarcinom-Zelllinien
(2006) Roser, Silvia; Rechkemmer, Gerhard
Colon cancer is the second most common cancer in Germany. The role of dietary fibre in the prevention of colon cancer is still controversial: Promising results from in vitro and animal studies are contradictory to inconsistent results from epidemiological stu-dies. Functional carbohydrates as constituents of prebiotic food can modify the colonic microflora for the benefit of short chain fatty acid (SCFA)-producing microbial strains. The SCFA-concentrations should also be increased in the distal part of the colon where most colon carcinomas are developing. SCFA are considered to be preventive against colon cancer. For this study, three different new functional oligosaccharides (OS, made of Isomaltulose and resistant starch) were produced from the Südzucker company and fermented in vitro with human feces of healthy test subjects. The resulting fermentation supernatants (FS) were tested in a cell culture system, using colon carcinoma cell lines of various degrees of differentiation (HT29, HT29 Clone 19A, T84). Cytotoxicity, proliferation, the induction of apoptosis, influences on the cell cycle and electrophysiological parameters were measured. Spectral photometric and flow cytometric methods were performed, as well as measurements in vertical diffusion chambers (Ussing chambers). The parallel testing of SCFA-mixtures with the same SCFA-concentrations as in the FS was included, as well as the testing of a FS ?Control? which was produced without OS-fermentation. Several independent fermentations revealed reproducible results regarding the SCFA-concentrations of the FS. After OS-fermentation, the ratio of the three major SCFA in the FS, acetate, propionate, and butyrate, was similar to that observed in vivo. The FS and SCFA-mixtures tested had a cytotoxic effect on all cell lines at the con-centration of 50 %. A dose dependent decrease in cell proliferation could be found, as well as the induction of apoptosis at a concentration of 50 %. Parallel testing of the analogous SCFA-mixtures showed that cytotoxic and proliferation inhibiting effects of the FS could be primarily attributed to their SCFA-content. This could not be confirmed for apoptosis induction: the SCFA-mixtures were mostly able to induce a higher apoptosis rate than the FS. Similarly, the effects of FS and SCFA-mixtures on the cell cycle were different: The SCFA-mixtures showed more potent inhibition of DNA-synthesis than the analogous FS, which generally led to an arrest in the G2-phase of the cell cycle. Neither FS nor SCFA-mixtures had an impact on transepithelial resistance or short circuit current of differentiated cell monolayers in Ussing chambers. The difference in the fermentation patterns of the various FS and the SCFA-concentrations of the SCFA-mixtures was not great enough to achieve significantly different results in the test systems used. Also, the various differentiation grades of the cell lines showed inconsistent results after treatment with FS and their SCFA-mixtures, so that no correlation could be found between degree of differentiation and test compound action. This study shows that the in vitro fermentation of OS with human feces results in reproducible SCFA-patterns in the FS, similar to the in vivo situation. For the screening of FS and their SCFA-mixtures, respectively, a spectrum of methods was established for the incubation with colon carcinoma cell lines of various differentiation states and of all stages of growth (exponential, subconfluent, confluent, fully differentiated monolayer). Indeed, the effects measured after incubation with FS could only in part been ascribed to their SCFA content. Other FS components than SCFA that play a role, especially regarding to their apoptosis inhibiting and cell cycle influencing effects, remain to be identified. Also, this study allows no conclusions to be drawn, which of the fermented OS is more promising in it?s beneficial influence on colon cancer preventing factors, e.g. the induction of apoptosis, than the other. Future studies should investigate FS with greater differences in their SCFA-concentrations. The same OS which were used for the in vitro fermentation, should also be tested in animal studies and human intervention studies to elucidate their fermentation patterns in vivo.
Mechanisms of frost adaptation and freeze damage in grapevine buds
(2002) Badulescu Valle, Radu Virgil; Blaich, Rolf
Mechanisms of frost hardening in compound (latent) buds of the grapevine cultivar ?Bacchus? were tested with different methods during three winters. The investigated parameters were LTE/HTE (low temperature exotherm/high temperature exotherm), water content, starch, sugar- and anions combination and bud histology. Water content from wood and buds was determined regularly every 2 weeks from March 1998 until Mai 2000. The lowest water content in wood and buds (about 40 %) was found between November and February. In general shoot sections and buds from the apical shoot area contained less water than in the basal area. Sugars and anions were analyzed with HPLC. The highest concentrations of soluble sugars were found in basal buds of the shoot, the lowest concentration in buds of the apical shoot area. Sucrose was the predominant soluble sugar, it was accompanied by glucose, fructose, sucrose, raffinose, and also stacchyose which was hitherto not described for grapevine buds. The concentration of soluble sugars increased during autumn and reached its maximum (around 150 mg/g dry matter) in November/December until the beginning of January then it decreased again to around 30 mg/g at bud burst. The predominant anion was sulphate while chloride could be detected only in traces. The anions reached their maximum at the beginning of January and in mid April. To evaluate the exotherm measuring method, model experiments were carried out with water drops (1µl) on filter paper and with small plant parts (leaf, stems, flower parts). Both the plant parts and the destilled water on the cellulose fiber freeze mainly between ?8 and ?15°C (an influence of the low osmotic value of the plant sap could not be found). After the first freezing the specimen were thawed and freezing repeated. The freezing points of the first and the second freezing cycle were significantly correlated. This shows that freezing does not occur at random, but is determined by ice nucleation sites characteristic for each sample. These sites even survive the physical destruction of the cells by the ice cristals. Further model experiments were carried out to get indications on possible barriers to ice cristal growth in plant tissue. Exotherm analysis was used to determine the freezing point of grapevine buds which is accompanied by a transient temperature rise called exotherm. The grapevine buds show 2 or more exotherms, one or two HTEs (high temperature exotherms) between ? 5 °C and ?10°C and the LTE (low temperature exotherm, sometimes more than one ) between ?10°C and ?25°C depending on the frost adaption of the buds. The HTEs are assumed to indicate the freezing of surface water or apoplastic water in the subtending tissue (bud pad), whereas the LTE (or LTEs) seem to be caused by freezing of the primary (and secondary) buds (shoot primordiy of the compound bud). The temperature minimum of the LTEs (down to ? 25 °C) is reached in January/February and is not influenced by humidity which, however, changes the THE values occuring usually around ? 10 ° and ? 4 °C, which are influenced by water in the bud scales. The LTEs of the buds in the lower area of the shoot were higher as compared to the buds in the middle and upper area of the shoot. The LTE analysis clearly shows the frost adaptation of the latent buds which usually reaches a maximum by the end of January but a clear relation to the changing air temperatures could not be established. Histological and cytological analyses were used to test for frost damage in bud parts and for changes during the cold adaptation. A modified staining method was developed to differentiate the cells. During automn and winter the buds contained a lot of starch grains which dissolved at bud burst. A permeability barrier between bud pad and shoot primordia could not be found, however it could be directly shown, that a HTE causes no cell damage in the buds, while after the appearence of the LTE(s) a disintegration of protoplasts in primary and secondary buds could be found. This is a direct evidence that LTEs indicates the death of the eyes in the complex grapevine bud. If after the appearance of the HTE the buds were held one day at this temperature before further cooling, no LTEs would appear. This and similar observations during the frost storage of grapevine cuttings is discussed in terms of the (harmless) ice formation in the bud base at moderate minus temperatures which would result in a freeze drying effect due to the lower water potential of the bud pad (in comparison to the non frozen eyes) and a further increase of the frost resistance of the growing points. If frost adapted grapevine shoots from the field were kept at 20°C deacclimation occurred after about 10 days. Accidentally wetted buds showed exotherms above ?4°C. In these buds and the watering water ice nucleating bacteria (Pseudomonas fluorescens) could be found.