No abstract

Mutants in the PRT1 gene of Arabidopsis thaliana are impaired in the degradation of a normally shortlived intracellular protein that contains a destabilizing Nterminal residue. Proteins bearing such residues are the substrates of an ubiquitin-dependent proteolytic system called the N-end rule pathway. The chromosomal position of PRT1 was determined, and the PRT1 gene was isolated by map-based cloning. The 45-kDa PRT1 protein contains two RING finger domains and one ZZ domain. No other proteins in databases match these characteristics of PRT1. There is, however, a weak similarity to Rad18p of Saccharomyces cerevisiae. The RING finger domains have been found in a number of other proteins that are involved in ubiquitin conjugation, consistent with the proposed role of PRT1 in the plant N-end rule pathway.

Pharmacological studies support the idea that nitric oxide (NO) serves as a retrograde messenger during long-term potentiation (LTP) in area CA1 of the hippocampus. Mice with a defective form of the gene for neuronal NO synthase (nNOS), however, exhibit normal LTP. The myristoyl protein endothelial NOS (eNOS) is present in the dendrites of CA1 neurons. Recombinant adenovirus vectors containing either a truncated eNOS (a putative dominant negative) or an eNOS fused to a transmembrane protein were used to demonstrate that membrane-targeted eNOS is required for LTP. The membrane localization of eNOS may optimally position the enzyme both to respond to Ca2+ influx and to release NO into the extracellular space during LTP induction.

Background:The intravasation of breast cancer into the lymphendothelium is an early step of metastasis. Little is known about the mechanisms of bulky cancer invasion into lymph ducts.Methods:To particularly address this issue, we developed a 3-dimensional co-culture model involving MCF-7 breast cancer cell spheroids and telomerase-immortalised human lymphendothelial cell (LEC) monolayers, which resembles intravasation in vivo and correlated the malignant phenotype with specific protein expression of LECs.Results:We show that tumour spheroids generate ‘circular chemorepellent-induced defects' (CCID) in LEC monolayers through retraction of LECs, which was induced by 12(S)-hydroxyeicosatetraenoic acid (HETE) secreted by MCF-7 spheroids. This 12(S)-HETE-regulated retraction of LECs during intravasation particularly allowed us to investigate the key regulators involved in the motility and plasticity of LECs. In all, 12(S)-HETE induced pro-metastatic protein expression patterns and showed NF-κB-dependent up-regulation of the mesenchymal marker protein S100A4 and of transcriptional repressor ZEB1 concomittant with down-regulation of the endothelial adherence junction component VE-cadherin. This was in accordance with ∼50% attenuation of CCID formation by treatment of cells with 10 μ Bay11-7082. Notably, 12(S)-HETE-induced VE-cadherin repression was regulated by either NF-κB or by ZEB1 since ZEB1 siRNA knockdown abrogated not only 12(S)-HETE-mediated VE-cadherin repression but inhibited VE-cadherin expression in general.Interpretation:These data suggest an endothelial to mesenchymal transition-like process of LECs, which induces single cell motility during endothelial transmigration of breast carcinoma cells. In conclusion, this study demonstrates that the 12(S)-HETE-induced intravasation of MCF-7 spheroids through LECs require an NF-κB-dependent process of LECs triggering the disintegration of cell–cell contacts, migration, and the generation of CCID.

To assess the value of anti-isocitrate dehydrogenase 1 (IDH1) immunohistochemistry for evaluating diffuse gliomas, we analyzed anti-IDH1-R132H immunohistochemistry using monoclonal antibodies DIA-H09 and IMab-1 and IDH1 gene sequencing in formalin-fixed and paraffin-embedded biopsy samples of 95 diffuse gliomas. We found concordant immunostaining results using the 2 antibodies in 94 (98.9%) of the 95 cases, but DIA-H09 generally showed a higher signal-to-background ratio than IMab-1 did. Fifty-five percent of cases showed anti-IDH1-R132H immunostaining of virtually all tumor cells and 15% of only a fraction of tumor cells. All cases with complete or partial immunostaining of the tumor tissue carried the IDH1-R132H mutation. In all cases with negative immunostaining results (approximately 30%), genetic analysis showed IDH1 wild-type or non-R132H-IDH1 mutations. In a single tiny biopsy, both anti-IDH1-R132H antibodies showed immunoreactivity, but genetic testing was inconclusive. Our data confirm anti-IDH1-R132H immunostaining as a reliable method for evaluation of IDH1 gene mutation status. They also suggest the following: (i) in some cases, nonspecific background staining or regional heterogeneity of IDH1-R132H protein expression may necessitate confirmatory genetic analysis; (ii) for individual cases, anti-IDH1-R132H immunostaining may not reliably identify infiltrating tumor cells admixed with preexisting or reactive glial cells; and (iii) in tiny biopsies, immunohistochemistry may be more sensitive for detection of IDH1-R132H mutation than genetic analysis.

The gene PRT1 of Arabidopsis, encoding a 45-kD protein with two RING finger domains, is essential for the degradation of F-dihydrofolate reductase, a model substrate of the N-end rule pathway of protein degradation. We have determined the function of PRT1 by expression in yeast (Saccharomyces cerevisiae). PRT1 can act as a ubiquitin protein ligase in the heterologous host. The identified substrates of PRT1 have an aromatic residue at their amino-terminus, indicating that PRT1 mediates degradation of N-end rule substrates with aromatic termini but not of those with aliphatic or basic amino-termini. Expression of model substrates in mutant and wild-type plants confirmed this substrate specificity. A ligase activity exclusively devoted to aromatic amino-termini of the N-end rule pathway is apparently unique to plants. The results presented also imply that other known substrates of the plant N-end rule pathway are ubiquitylated by one or more different ubiquitin protein ligases.

Background:Many cancers spread through lymphatic routes, and mechanistic insights of tumour intravasation into the lymphatic vasculature and targets for intervention are limited. The major emphasis of research focuses currently on the molecular biology of tumour cells, while still little is known regarding the contribution of lymphatics.Methods:Breast cancer cell spheroids attached to lymphendothelial cell (LEC) monolayers were used to investigate the process of intravasation by measuring the areas of ‘circular chemorepellent-induced defects' (CCID), which can be considered as entry gates for bulky tumour intravasation. Aspects of tumour cell intravasation were furthermore studied by adhesion assay, and siRNA-mediated knockdown of intracellular adhesion molecule-1 (ICAM-1). Replacing cancer spheroids with the CCID-triggering compound 12(S)-hydroxyeicosatetraenoic acid (HETE) facilitated western blot analyses of Bay11-7082- and baicalein-treated LECs.Results:Binding of LECs to MCF-7 spheroids, which is a prerequisite for CCID formation, was mediated by ICAM-1 expression, and this depended on NF-κB and correlated with the expression of the prometastatic factor S100A4. Simultaneous inhibition of NF-κB with Bay11-7082 and of arachidonate lipoxygenase (ALOX)-15 with baicalein prevented CCID formation additively.Conclusion:Two mechanisms contribute to CCID formation: ALOX15 via the generation of 12(S)-HETE by MCF-7 cells, which induces directional migration of LECs, and ICAM-1 in LECs under control of NF-κB, which facilitates adhesion of MCF-7 cells to LECs.

In our study, the application of microarray analysis in prenatal testing proved to be a valuable tool for the identification of submicroscopic chromosomal aberrations where conventional cytogenetic methods failed. Selection of appropriate resolution was found to be critical to obtain reliable, diagnostically conclusive data.