Aptamers containing thymidine 3'-O-phosphorodithioates: synthesis and binding to nuclear factor-kappaB

Aptamers targeting NF-kappaB containing thymidine 3'-O-phosphorodithioates in selected positions of an oligonucleotide duplex were synthesized. Binding affinities to NF-kappaB varied with the number and positions of the dithioate backbone substitutions. One of the aptamers showed specific binding to a single NF-kappaB dimer in cell culture extracts.     

Trichome cell growth in Arabidopsis thaliana can be derepressed by mutations in at least five genes

Leaf trichomes in Arabidopsis are unicellular epidermal hairs with a branched morphology. They undergo successive endoreduplication rounds early during cell morphogenesis. Mutations affecting trichome nuclear DNA content, such as triptychon or glabra3, alter trichome branching. We isolated new mutants with supernumerary trichome branches, which fall into three unlinked complementation groups: KAKTUS and the novel loci, POLYCHOME and RASTAFARI. They map to chromosomes IV, II, and V, respectively. The trichomes of these mutants presented an increased DNA content, although to a variable extent. The spindly-5 mutant, which displays a constitutive gibberellin response, also produces overbranched trichomes containing more nuclear DNA. We analyzed genetic interactions using double mutants and propose that two independent pathways, defined by SPINDLY and TRIPTYCHON, act to limit trichome growth. KAKTUS and POLYCHOME might have redundant actions mediating gibberellin control via SPINDLY. The overall leaf polysomaty was not notably affected by these mutations, suggesting that they affect the control of DNA synthesis in a tissue- or cell type-specific manner. Wild-type tetraploids also produce overbranched trichomes; they displayed a shifted polysomaty in trichomes and in the whole leaf, suggesting a developmental program controlling DNA increases via the counting of endoreduplication rounds.     

Gangliosides are Transported from the Plasma Membrane to Intralysosomal Membranes as Revealed by Immuno-Electron Microscopy

A biotin-labeled derivative of the ganglioside GM1 (biotin-GM1) was used to study its transport along the endocytic pathway of cultured fibroblasts by immuno-electron microscopy. Using electron dense endocytic tracers we could demonstrate that late endosomes and lysosomes of these cells are long living organelles with a high content of internal membranes. Our studies show that during endocytosis the biotin-GM1 was transported to these intraendosomal and intralysosomal membranes. These observations support the hypothesis that glycosphingolipids (GSL) are preferentially degraded in intralysosomal vesicles.     

Mutations in the pilz group genes disrupt the microtubule cytoskeleton and uncouple cell cycle progression from cell division in Arabidopsis embryo and endosperm

Organised cell division and expansion play important roles in plant embryogenesis. To address their cellular basis, we have analysed Arabidopsis abnormal-embryo mutants which were isolated for their characteristic phenotype: mutant embryos are small, mushroom-shaped ("pilz") and consist of only one or few large cells each containing one or more variably enlarged nuclei and often cell wall stubs. These 23 mutants represent four genes, PFIFFERLING, HALLIMASCH, CHAMPIGNON, and PORCINO, which map to different chromosomes. All four genes have very similar mutant phenotypes although porcino embryos often consisted of only one large cell. The endosperm did not cellularise and contained a variably reduced number of highly enlarged nuclei. By contrast, genetic evidence suggests that these genes are not required for gametophyte development. Expression of cell cycle genes, Cdc2a, CyclinA2 and CyclinB1, and the cytokinesis-specific KNOLLE gene was not altered in mutant embryos. However, KNOLLE syntaxin accumulated in patches but no KNOLLE-positive structure resembling a forming cell plate occurred in mitotic cells. A general defect in microtubule assembly was observed in all mutants. Interphase cells lacked cortical microtubules, and spindles were absent from mitotic nuclei although in rare cases, short stubs of microtubules were attached to partially condensed chromosomes. Our results suggest that the cellular components affected by the pilz group mutations are necessary for continuous microtubule organisation, mitotic division and cytokinesis but do not mediate cell cycle progression.     

Endoplasmic reticulum-associated degradation: exceptions to the rule

Quality control mechanisms in the endoplasmic reticulum (ER) ensure that misfolded proteins are recognized and targeted for degradation. According to the current view of ER-associated degradation (ERAD), the degradation does not occur in the ER itself but requires the retrotranslocation of the proteins to the cytosol where they are degraded by proteasomes. Although this model appears to be valid for many different proteins a number of exceptions from this rule suggest that additional proteasome-independent ERAD pathways may exist. In this review, we will summarize what is known about these alternative ERAD pathways.     

Cell migration: mechanisms of rear detachment and the formation of migration tracks

Cell migration is central to many biological and pathological processes, including embryogenesis, tissue repair and regeneration as well as cancer and the inflammatory response. In general, cell migration can be usefully conceptualized as a cyclic process. The initial response of a cell to a migration-promoting agent is to polarize and extend protrusions in the direction of migration. These protrusions can be large, broad lamellipodia or spike-like filopodia, are usually driven by actin polymerization, and are stabilized by adhering to the extracellular matrix (ECM) via transmembrane receptors of the integrin family linked to the actin cytoskeleton. These adhesions serve as traction sites for migration as the cell moves forward over them, and they must be disassembled at the cell rear, allowing it to detach. The mechanisms of rear detachment and the regulatory processes involved are not well understood. The disassembly of adhesions that is required for detachment depends on a coordinated interaction of actin and actin-binding proteins, signaling molecules and effector enzymes including proteases, kinases and phosphatases. Originally, the biochemically regulated processes leading to rear detachment of migrating cells were thought not to be necessarily accompanied by any loss of cell material. However, it has been shown that during rear detachment long tubular extensions, the retracting fibers, are formed and that "membrane ripping" occurs at the cell rear. By this process, a major fraction of integrin-containing cellular material is left behind forming characteristic migration tracks that exactly mark the way a cell has taken.     

Biological roles of APP in the epidermis

The amyloid precursor protein (APP) was initially detected in cells of the central nervous system where it is considered to be involved in the pathogenesis of Alzheimer's disease. However, APP is also found in peripheral organs with exceptionally strong expression in the mammalian epidermis where it fulfils a variety of distinct biological roles. Full length APP appears to facilitate keratinocyte adhesion due to its ability to interact with the extracellular matrix. The C-terminus of APP also serves as adapter protein for binding the motor protein kinesin thereby mediating the centripetal transport of melanosomes in epidermal melanocytes. By the action of alpha-secretase sAPPalpha, the soluble N-terminal portion of APP, is released. sAPPalpha has been shown to be a potent epidermal growth factor thus stimulating proliferation and migration of keratinocytes as well as the exocytic release of melanin by melanocytes. The release of sAPPalpha can be almost completely blocked by inhibiting alpha-secretase with hydroxamic acid-based zinc metalloproteinase inhibitors. In hyperproliferative keratinocytes from psoriatic skin this inhibition results in normalized growth.     

Cytoprotective function of sAppalpha in human keratinocytes

sAPPalpha, the soluble form of the beta-amyloid precursor protein, has been shown to act as a potent epidermal growth factor by stimulating keratinocyte proliferation and migration. In this report we provide evidence for a cytoprotective role of sAPPalpha. As a model we used HaCaT cells and normal human keratinocytes (NHK) cultured in the absence of fetal calf serum and bovine pituitary extract. Under these conditions keratinocytes began to undergo apoptosis at increasing rates after 96 h of culture. Surprisingly, keratinocytes were protected from apoptosis by the addition of 50 nM recombinant sAPPalpha. Subsequent experiments were performed to elucidate the regulatory basis of the cytoprotective role of sAPPalpha. We found that recombinant sAPPalpha facilitated the substrate adhesion of keratinocytes in the first 30 minutes after seeding. The basis for this adhesion-promoting function was shown by the ability of recombinant sAPPalpha to continuously coat the culture dish thereby promoting the ability to bind keratinocytes. A second mechanism explaining the cytoprotective role was found in the significant inhibition of apoptosis by recombinant sAPPalpha. In HaCaT cells moderate UV-B irradiation was sufficient to induce apoptosis. In contrast, induction of apoptosis in NHK required additionally the depletion of endogenous sAPPalpha suggesting that sAPPalpha mediates protection against UV-B irradiation. Staurosporine-induced apoptosis rates were significantly reduced by about 59% after addition of recombinant sAPPalpha. These results show that sAPPalpha exerts a pronounced cytoprotective effect and that this effect is mediated by facilitated cell adhesion and by the antiapoptotic function of sAPPalpha.     

Protein expression profiling identifies molecular targets of quercetin as a major dietary flavonoid in human colon cancer cells

A high dietary intake of plant foods is thought to contribute to the prevention of colorectal cancers in humans and flavonoids as part of such a diet are considered to contribute to those protective effects. Quercetin is a major dietary flavonoid consumed with a diet rich in onions, tea, and apples. We used HT-29 human colon cancer cells and investigated the effects of quercetin on proliferation, apoptosis, and differentiation as processes shown to be disregulated during cancer development. To identify the cellular targets of quercetin action, two-dimensional gel electrophoresis was performed and proteins altered in expression level after quercetin exposure of cells were identified by mass spectrometry of peptide fragments generated by tryptic digestion. Quercetin inhibited the proliferation of HT-29 cells with an IC(50)-value of 81.2 +/- 6.6 microM. Cell differentiation based on surface expression of alkaline phosphatase was enhanced 4-fold and the activity of the pro-apoptotic effector caspase-3 increased 3-fold. Those effects were associated with the regulation of heat-shock proteins and annexins shown to both play a crucial role in the process of apoptosis. Cytoskeletal caspase substrates were found as regulated as well and various proteins involved in intermediary metabolism and in gene regulation showed altered steady-state expression levels upon quercetin treatment of cells. In conclusion, quercetin alters the levels of a variety of proteins involved in growth, differentiation, and apoptosis of colon cancer cells. Their identification as molecular targets of quercetin may explain the anti-cancer activities of this flavonoid.     

The anaphase promoting complex/cyclosome is recruited to centromeres by the spindle assembly checkpoint

The anaphase promoting complex/cyclosome (APC/C) is crucial to the control of cell division (for a review, see ref. 1). It is a multi-subunit ubiquitin ligase that, at defined points during mitosis, targets specific proteins for proteasomal degradation. The APC/C is itself regulated by the spindle or kinetochore checkpoint, which has an important role in maintaining genomic stability by preventing sister chromatid separation until all chromosomes are correctly aligned on the mitotic spindle. The spindle checkpoint regulates the APC/C by inactivating Cdc20, an important co-activator of the APC/C. There is also evidence to indicate that the spindle checkpoint components and Cdc20 are spatially regulated by the mitotic apparatus, in particular they are recruited to improperly attached kinetochores. Here, we show that the APC/C itself co-localizes with components of the spindle checkpoint to improperly attached kinetochores. Indeed, we provide evidence that the spindle checkpoint machinery is required to recruit the APC/C to kinetochores. Our data indicate that the APC/C could be regulated directly by the spindle checkpoint.