Targeting Homologous Recombination in Notch-Driven C. elegans Stem Cell and Human Tumors

Mammalian NOTCH1-4 receptors are all associated with human malignancy, although exact roles remain enigmatic. Here we employ glp-1(ar202), a temperature-sensitive gain-of-function C. elegans NOTCH mutant, to delineate NOTCH-driven tumor responses to radiotherapy. At ≤20°C, glp-1(ar202) is wild-type, whereas at 25°C it forms a germline stem cell⁄progenitor cell tumor reminiscent of human cancer. We identify a NOTCH tumor phenotype in which all tumor cells traffic rapidly to G2⁄M post-irradiation, attempt to repair DNA strand breaks exclusively via homology-driven repair, and when this fails die by mitotic death. Homology-driven repair inactivation is dramatically radiosensitizing. We show that these concepts translate directly to human cancer models.     

Unsaturated oligogalacturonic acids are generated by in vitro treatment of pectin with human faecal flora.

Pectins with a degree of esterification (DE) of 95, 66, 34 and 0%, respectively, were incubated in vitro with human faecal flora (pH 7.8). The concentration and composition of oligogalacturonic acids (oligoGalA) generated were determined using high-performance thin-layer chromatography (HPTLC) with UV and colorimetric detection. In the first period of the anaerobic degradation, the pectin macromolecules were fragmented into unsaturated oligoGalA as intermediate products by the action of bacterial pectate lyases. Depending on the incubation time and the DE of pectin, the amount of unsaturated oligoGalA having different degrees of polymerization changed continuously. These oligoGalA were present in the cultures for some hours. Mixtures of unsaturated di-, tri- and tetraGalA were the end products of a pectate lyase action. Later, the oligoGalA disappear as a result of their further fermentation by the gastrointestinal microflora under formation of short-chain fatty acids (SCFA). Low-esterified pectins were depolymerized and fermented faster than the highly esterified by the human faecal flora in vitro. Furthermore, a mixture of unsaturated oligoGalA prepared from pectic acid by the action of pectate lyase from Erwinia carotovora was completely fermented by human faecal flora.     

The biometry of gills of 0-group European flounder

The gill surface area of 0-group, post-metamorphic Pleuronectes flesus L. was examined using digital image analysis software and expressed in relation to body mass according to the equation log Y=loga+c logW ( a =239·02; c =0·723). The components that constitute gill area, total filament length, interlamellar space and unilateral lamellar area were measured. The measurement of the length of every filament on all eight arches showed that commonly used methods of calculation can lead to an under-estimation of up to 24% of total filament length. Direct measurements of unilateral lamellar area with digital image analysis showed that previously reported gill area data for the same species was over-estimated by as much as 58%. In addition, in this species the neglect of gill pouch asymmetry after metamorphosis, can bring about a 14% over-estimation of total gill area.     

Determination of turnover of androstenedione to estrone via aromatase in estrogen receptor positive and negative human breast cancer cell lines [Poster 48]

In one estrogen receptor (ER) negative (MDA-MB-231) and two ER positive human breast cancer cell lines (T-47-D,SK-BR-3) we measured aromatase activity by [³H]water assay and estrone (E1) production by thin-layer chromatography. Compared with ether extraction and charcoal method, lyophilization proved to be the most sensitive technique to measure the quantity of [³H]water. The extremely low contamination of the water soluble phase by [1ß-³H]androstenedione (0.02%), as well as the lack of errors due to conjugated steroids, offers the possibility to measure changes of cellular aromatase activity even at very low levels. In contrast to SK-BR-3 and MDA-MB-231 cells, we found no aromatase activity in T-47-D cells. There was no coincidence between ER status and aromatase activity. Proliferation of tumor cells was parallel with a continuous increase of aromatase activity and E1 production during mitogenic growth phase reaching highest levels at the transition from log to plateau-phase.     

Mitochondrial precursor proteins are imported through a hydrophilic membrane environment

We have analyzed how translocation intermediates of imported mitochondrial precursor proteins, which span contact sites, interact with the mitochondrial membranes. F1-ATPase subunit beta (F1 beta) was trapped at contact sites by importing it into Neurospora mitochondria in the presence of low levels of nucleoside triphosphates. This F1 beta translocation intermediate could be extracted from the membranes by treatment with protein denaturants such as alkaline pH or urea. By performing import at low temperatures, the ADP/ATP carrier was accumulated in contact sites of Neurospora mitochondria and cytochrome b2 in contact sites of yeast mitochondria. These translocation intermediates were also extractable from the membranes at alkaline pH. Thus, translocation of precursor proteins across mitochondrial membranes seems to occur through an environment which is accessible to aqueous perturbants. We propose that proteinaceous structures are essential components of a translocation apparatus present in contact sites.     

Structural analysis of 5S rRNA, 5S rRNA-protein complexes and ribosomes employing RNase H and d(GTTCGG)

The hybridization of d(GTTCGG) to eubacterial 5S rRNAs, 5S rRNA-protein complexes, 70S ribosomes and 50S and 30S ribosomal subunits was investigated. This oligonucleotide, which may be considered to be an analogue of the T psi CG loop of tRNAs, was chosen in order to investigate a possible interaction between tRNAs with ribosomal components during protein synthesis. The hybridization was analysed by RNase H hydrolysis studies and, in the case of the ribosomes and ribosomal subunits, in addition with the radioactively labelled oligodeoxyribonucleotide in binding studies. The results obtained lead to the conclusion that nucleotides in loop c, i.e. positions 42-47, are available for oligonucleotide interaction in free Escherichia coli and Bacillus stearothermophilus 5S rRNAs and not available in the corresponding 5S rRNA-protein complexes. The 70S ribosomes and ribosomal subunits did not interact with the oligonucleotide. Under the assumption that d(GTTCGG) is an analogue of the T psi CG loop of tRNAs and in view of the results obtained, we conclude that in the unprogrammed ribosomes the T psi CG loop of tRNAs does not interact via standard Watson-Crick base pairs with the ribosomal 5S, 16S or 23S RNAs.