CD95 death-inducing signaling complex formation and internalization occur in lipid rafts of type I and type II cells

We investigated the membrane localization of CD95 in type I and type II cells, which differ in their ability to recruit and activate caspase-8. We found that CD95 was preferentially located in lipid rafts of type I cells, while it was present both in raft and non-raft plasma membrane sub-domains of type II cells. After stimulation, CD95 located in phospholipid-rich plasma membrane was recruited to lipid rafts in both types of cells. Similarly, CD95 cross-linking resulted in caspase-independent translocation of FADD/MORT1 and caspase-8 to the lipid rafts, which was prevented by a death domain-defective receptor. CD95 internalization was then rapid in type I and delayed in type II cells and showed a substantial correlation with the kinetics of Fas-associated death domain (FADD)and caspase-8 recruitment to lipid rafts. Finally, electron microscopy analysis showed that after CD95 stimulation lipid rafts aggregated in large clusters that were internalized in endosomal vesicles, where caspase-8 underwent massive processing. Taken together, our data demonstrate that CD95 death-inducing signaling complex formation and internalization in type I and type II cells occur in lipid rafts, which are a major site of caspase-8 activation.     

Frequency of RET mutations in long- and short-segment Hirschsprung disease

Hirschsprung disease, or congenital aganglionic megacolon, is a genetic disorder of neural crest development affecting 1:5,000 newborns. Mutations in the RET proto-oncogene, repeatedly identified in the heterozygous state in both long- and short-segment Hirschsprung patients, lead to loss of both transforming and differentiating capacities of the activated RET through a dominant negative effect when expressed in appropriate cellular systems. The approach of single-strand conformational polymorphism analysis established for all the 20 exons of the RET proto-oncogene, and previously used to screen for point mutations in Hirschsprung patients allowed us to identify seven additional mutations among 39 sporadic and familial cases of Hirschsprung disease (detection rate 18%). This relatively low efficiency in detecting mutations of RET in Hirschsprung patients cannot be accounted by the hypothesis of genetic heterogeneity, which is not supported by the results of linkage analysis in the pedigrees analyzed so far. Almost 74% of the point mutations in our series, as well as in other patient series, were identified among long segment patients, who represented only 25% of our patient population. The finding of a C620R substitution in a patient affected with total colonic aganglionosis confirms the involvement of this mutation in the pathogenesis of different phenotypes (i.e., medullary thyroid carcinoma and Hirschsprung). Finally the R313Q mutation identified for the first time in homozygosity in a child born of consanguineous parents is associated with the most severe Hirschsprung phenotype (total colonic aganglionosis with small bowel involvement). Hum Mutat 9:243–249, 1997. © 1997 Wiley-Liss, Inc.     

Influence of Initial Temperature and Convective Heat Loss on the Self-Propagating Reaction in Al/Ni Multilayer Foils

A two-dimensional numerical model for self-propagating reactions in Al/Ni multilayer foils was developed. It was used to study thermal properties, convective heat loss, and the effect of initial temperature on the self-propagating reaction in Al/Ni multilayer foils. For model adjustments by experimental results, these Al/Ni multilayer foils were fabricated by the magnetron sputtering technique with a 1:1 atomic ratio. Heat of reaction of the fabricated foils was determined employing Differential Scanning Calorimetry (DSC). Self-propagating reaction was initiated by an electrical spark on the surface of the foils. The movement of the reaction front was recorded with a high-speed camera. Activation energy is fitted with these velocity data from the high-speed camera to adjust the numerical model. Calculated reaction front temperature of the self-propagating reaction was compared with the temperature obtained by time-resolved pyrometer measurements. X-ray diffraction results confirmed that all reactants reacted and formed a B2 NiAl phase. Finally, it is predicted that (1) increasing thermal conductivity of the final product increases the reaction front velocity; (2) effect of heat convection losses on reaction characteristics is insignificant, e.g., the foils can maintain their characteristics in water; and (3) with increasing initial temperature of the foils, the reaction front velocity and the reaction temperature increased.     

Inhibition of in vitro growth and arrest in the G0/G1 phase of HCT8 line human colon cancer cells by kaempferide triglycoside from Dianthus caryophyllus

The effects of phytoestrogens have been studied in the hypothalamic‐pituitary‐gonadal axis and in various non‐gonadal targets. Epidemiologic and experimental evidence indicates a protective effect of phytoestrogens also in colorectal cancer. The mechanism through which estrogenic molecules control colorectal cancer tumorigenesis could possibly involve estrogen receptor β, the predominantly expressed estrogen receptor subtype in colon mucosa. To validate this hypothesis, we therefore used an engineered human colon cancer cell line induced to overexpress estrogen receptor β, beside its native cell line, expressing very low levels of ERβ and not expressing ERα; as a phytoestrogenic molecule, we used kaempferide triglycoside, a glycosylated flavonol from a Dianthus caryophyllus cultivar. The inhibitory properties of this molecule toward vegetal cell growth have been previously demonstrated: however, no data on its activity on animal cell or information about the mechanism of this activity are available. Kaempferide triglycoside proved to inhibit the proliferation of native and estrogen receptor β overexpressing colon cancer cells through a mechanism not mediated by ligand binding dependent estrogen receptor activation. It affected HCT8 cell cycle progression by increasing the G₀/G₁ cell fraction and in estrogen receptor β overexpressing cells increased two antioxidant enzymes. Interestingly, the biological effects of this kaempferide triglycoside were strengthened by the presence of high levels of estrogen receptor β. Pleiotropic molecular effects of phytoestrogens may explain their protective activity against colorectal cancer and may represent an interesting area for future investigation with potential clinical applications. Copyright © 2010 John Wiley & Sons, Ltd.