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.     

Discriminatory Role of Detergent-Resistant Membranes in the Dimerization and Endocytosis of Prostate-Specific Membrane Antigen

Prostate-specific membrane antigen (PSMA) is a type-II membrane glycoprotein that was initially identified in LNCaP cells. It is expressed at elevated levels in prostate cancer. In view of the correlation between the expression levels of PSMA and disease grade and stage, PSMA is considered to be one of the most promising biomarkers in the diagnosis and treatment of prostate cancer. In LNCaP cells PSMA undergoes internalization via clathrin-coated pits followed by accumulation in the endosomes. PSMA associates with different types of detergent-resistant membranes (DRMs) along the secretory pathway. Its mature form is mainly insoluble in Lubrol WX, but does not associate with Triton X-100-DRMs. To understand the mechanism of PSMA internalization we investigated its association during internalization with DRMs. For this purpose, internalization was induced by antibody cross-linking. We demonstrate at the biochemical and cell biological levels that: [i] exclusively homodimers of PSMA are associated with Lubrol WX-DRMs, [ii] antibody-induced cross-linking of PSMA molecules results in a time-dependent partitioning into another DRMs type, namely Triton X-100-DRMs, and [iii] concomitant with its association with Triton-X-100-DRMs internalization of PSMA occurs along tubulin filaments. In a previous work (Colombatti et al. (2009) PLoS One 4: e4608) we demonstrated that the small GTPases RAS and RAC1 and the MAPKs p38 and ERK1/2 are activated during antibody cross-linking. As downstream effects of this activation we observed a strong induction of NF-kB associated with an increased expression of IL-6 and CCL5 genes and that IL-6 and CCL5 enhanced the proliferative potential of LNCaP cells synergistically. These observations together with findings reported here hypothesize a fundamental role of DRMs during activation of PSMA as platforms for trafficking, endocytosis and signalling. Understanding these mechanisms constitutes an essential prerequisite for utilization of PSMA as a therapeutically suitable target in prostate cancer.     

Longitudinal Metabolomic Profiling of Amino Acids and Lipids across Healthy Pregnancy

Pregnancy is characterized by a complexity of metabolic processes that may impact fetal development and ultimately, infant health outcomes. However, our understanding of whole body maternal and fetal metabolism during this critical life stage remains incomplete. The objective of this study is to utilize metabolomics to profile longitudinal patterns of fasting maternal metabolites among a cohort of non-diabetic, healthy pregnant women in order to advance our understanding of changes in protein and lipid concentrations across gestation, the biochemical pathways by which they are metabolized and to describe variation in maternal metabolites between ethnic groups. Among 160 pregnant women, amino acids, tricarboxylic acid (TCA) cycle intermediates, keto-bodies and non-esterified fatty acids were detected by liquid chromatography coupled with mass spectrometry, while polar lipids were detected through flow-injected mass spectrometry. The maternal plasma concentration of several essential and non-essential amino acids, long-chain polyunsaturated fatty acids, free carnitine, acetylcarnitine, phosphatidylcholines and sphingomyelins significantly decreased across pregnancy. Concentrations of several TCA intermediates increase as pregnancy progresses, as well as the keto-body β-hydroxybutyrate. Ratios of specific acylcarnitines used as indicators of metabolic pathways suggest a decreased beta-oxidation rate and increased carnitine palmitoyltransferase-1 enzyme activity with advancing gestation. Decreasing amino acid concentrations likely reflects placental uptake and tissue biosynthesis. The absence of any increase in plasma non-esterified fatty acids is unexpected in the catabolic phase of later pregnancy and may reflect enhanced placental fatty acid uptake and utilization for fetal tissue growth. While it appears that energy production through the TCA cycle increases as pregnancy progresses, decreasing patterns of free carnitine and acetylcarnitine as well as increased carnitine palmitoyltransferase-1 rate and β-hydroxybutyrate levels suggest a concomitant upregulation of ketogenesis to ensure sufficient energy supply in the fasting state. Several differences in metabolomic profiles between Hispanic and non-Hispanic women demonstrate phenotypic variations in prenatal metabolism which should be considered in future studies.     

Cytogenetic study of F1 hybrids betweenCrepis dinarica andCrepis froelichiana (Asteraceae)

Crepis dinarica andC. froelichiana are two closely related species of theC. praemorsa complex. Even though they exhibit the same chromosome number (2n = 8) and similar idiogram shape, they differ widely in quantity and distribution of heterochromatin bands. The hybrids between these two species comprise three morphological types. Parental genomes were distinguished in hybrids by Giemsa differential staining (C-banding). Although meiosis presents only a few abnormalities (about 2.4%), the percentage of aborted pollen grains is very high (90%).     

DNA recovery and direct detection of Tn5 sequences from soil

Specific Tn5 sequences inserted in the genome of Enterobacter agglomerans were detected in EcoRI digested DNA directly recovered from soil 70 d after its inoculation with the bacteria, when these were no longer culturable on agar medium. A new method of DNA extraction from soil was used. No amplification of DNA sequences by PCR was needed.     

The effect of propranolol on whole-body microvibrations during examination stress

Whole-body microvibrations (MV) in three dimensions were measured in 51 volunteers, all medical students, 26 without and 25 with beta-receptor blockade (propranolol), immediately before a practical physiology examination and during the ensuing vacation. Propranolol impeded the increase in MV values in all three axes, significantly those in the z axis (vertical), the differences in MV values between the two measurements being minimal in the beta-receptor blocked group. On the other hand, propranolol enhanced MV in the x axis (anteroposterior) and the y axis (transverse), the y axis difference being significant only in females. Propranolol obviously relieves examination stress: the majority of candidates (52%) felt "quieter" in the examination with than in other similar situations without beta-receptor blockade. Propranolol was, however, without effect on the examination results. The rectified impulse in the z axis when related to body weight (Jz) correlates linearly with the calculated cardiac output. Propranolol, however, reduced cardiac output more than Jz, pointing to a Jz component non-sensitive to beta-receptor blockade. The part played by muscle tonus, mainly reflected in the y axis, thus remains unknown. The large and slow oscillations in the x and y axes, observed particularly in beta-receptor blocked females, might be attributed to diminution in standing ability.     

Bradyrhizobium japonicum mutants defective in root-nodule bacteroid development and nitrogen fixation

The genome of the slow-growing Bradyrhizobium japonicum (strain 110) was mutagenized with transposon Tn5. A total of 1623 kanamycin/streptomycin resistant derivatives were screened in soybean infection tests for nodulation (Nod) and symbiotic nitrogen fixation (Fix). In this report we describe 14 strains possessing a stable, reproducible Nod⁺Fix^- phenotype. These strains were also grown under microaerobic culture conditions to test them for free-living nitrogen fixation activity (Nif). In addition to strains having reduced Fix and Nif activities, there were also strains that had reduced symbiotic Fix activity but were Nif⁺ ex planta.Analysis of the genomic structure revealed that the majority of the strains had a single Tn5 insertion without any further apparent physical alteration. A few strains had additional insertions (by Tn5 or IS50), or a deletion, or had cointegrated part of the vector used for Tn5 mutagenesis. One of the insertions was found in a known nif gene (nifD) whereas all other mutations seem to affect different, hitherto unknown genes or operons.Several mutant strains had an altered nodulation phenotype, inducing numerous, small, widely distributed nodules. Light and electron microscopy revealed that most of these mutants were defective in different stages of bacteroid development and/or bacteroid persistence. The protein patterns of the mutants were inspected by two-dimensional gel electrophoresis after labelling microaerobic cultures with l-(³⁵S)methionine. Of particular interest were mutants lacking a group of proteins the synthesis of which was known to be under oxygen control. Such strains can be regarded as potential regulatory mutants.     

Binding sites for monoclonal antibodies and for mRNPs on SV40 large T-antigen determined with a cleavage map

Immune complexes of simian virus 40 large T-antigen with monoclonal papovavirus protein antibodies PAb 416, PAb 402, or PAb 423 were bound to protein-A-Sepharose and then cleaved into discrete fragments by limited tryptic proteolysis. PAb 402 protected a specific cleavage site, located approximately within amino acid residues 450-500, from tryptic proteolysis; PAb 423 protected another site within residues 675-699. As shown by immunoblotting, 125I-labeled PAb 416 was bound to a 17-kDa N-terminal fragment of large T-antigen (amino acid residues 1-130), and PAb 423 was bound to several overlapping fragments derived from the C terminus of large T-antigen. These monoclonal antibodies were then used as accessibility probes to study the interaction of mRNPs with cytoplasmic large T-antigen. Whereas small T-antigen and nuclear large T-antigen were fully immunoreactive, cytoplasmic large T-antigen reacted poorly with PAb 402 or polyclonal antibodies unless the mRNP moiety was removed by treatment with EDTA/RNase A. In contrast, mRNP/T-antigen complexes were fully immunoreactive with PAb 416 or PAb 423 and did not require treatment with EDTA/RNase A. The results suggest that the binding site of PAb 402 is blocked due to the interaction with mRNPs whereas the N-terminal binding site of PAb 416 and the C-terminal binding site of PAb 423 remain accessible to antibodies.     

Evaluation of Ia+ tumor cell lines and peritoneal exudate macrophages as accessory cells: differential requirements for the activation of certain T cell functions

Several Ia+ (BC3A, TA3, D1B) or Ia-inducible (WEHI-3, P388D1) tumor lines were tested for accessory cell function for the activation of antigen-specific T cell proliferation and for the induction of T helper cells that help B cells in antibody production. All lines were able to induce antigen-specific T cell proliferation in an MHC-restricted way, but none activated T helper cells to soluble antigens under all conditions tested. In comparison, starch-induced peritoneal exudate macrophages induced T cell proliferation as well as T cell help. Some of the lines tested induced nonspecific suppressor cells that were Ly-2-positive and partially or completely inhibited antibody responses. The induction of suppressor cells, however, is not the reason for the failure of the tumor lines to activate T helper cells. These data indicate that antigen-specific T cell proliferation and helper activity do not necessarily correlate.     

Antheseabhängige UV-Muster in Blütenständen von Asteraceen

The pseudanthia ofHeliopsis scabra andRudbeckia vulgaris (Asteraceae) were examined during the anthesis for differences in their UV patterns. Distinct changes in the reflectance and absorbance properties could be observed. The results suggest a close correlation between different stages of floral development and pollinator attraction.

Herrn Prof. Dr.Lothar Geitler zum 90. Geburtstag gewidmet.