Association Between the Rat Homologue of CO-029, a Metastasis-associated Tetraspanin Molecule and Consumption Coagulopathy

Recently, we have described a panel of metastasis-associated antigens in the rat, i.e., of molecules expressed on metastasizing, but not on nonmetastasizing tumor lines. One of these molecules, recognized by the monoclonal antibody D6.1 and named accordingly D6.1A, was found to be abundantly expressed predominantly on mesenchyme-derived cells. The DNA of the antigen has been isolated and cloned. Surprisingly, the gene product proved to interfere strongly with coagulation.

The 1.182-kb cDNA codes for a 235–amino acid long molecule with a 74.2% homology in the nucleotide and a 70% homology in the amino acid sequence to CO-029, a human tumor-associated molecule. According to the distribution of hydrophobic and hydrophilic amino acids, D6.1A belongs to the tetraspanin superfamily. Western blotting of D6.1A-positive metastasizing tumor lines revealed that the D6.1A, like many tetraspanin molecules, is linked to further membrane molecules, one of which could be identified as α6β1 integrin. Transfection of a low-metastasizing tumor cell line with D6.1A cDNA resulted in increased metastatic potential and provided a clue as to the functional role of D6.1A. We noted massive bleeding around the metastases and, possibly as a consequence, local infarctions predominantly in the mesenteric region and all signs of a consumption coagulopathy. By application of the D6.1 antibody the coagulopathy was counterregulated, though not prevented.

It has been known for many years that tumor growth and progression is frequently accompanied by thrombotic disorders. Our data suggest that the phenomenon could well be associated with the expression of tetraspanin molecules.

     

Functional evaluation of domain-domain interactions and human protein interaction networks

MOTIVATION: Large amounts of protein and domain interaction data are being produced by experimental high-throughput techniques and computational approaches. To gain insight into the value of the provided data, we used our new similarity measure based on the Gene Ontology (GO) to evaluate the molecular functions and biological processes of interacting proteins or domains. The applied measure particularly addresses the frequent annotation of proteins or domains with multiple GO terms. RESULTS: Using our similarity measure, we compare predicted domain-domain and human protein-protein interactions with experimentally derived interactions. The results show that our similarity measure is of significant benefit in quality assessment and confidence ranking of domain and protein networks. We also derive useful confidence score thresholds for dividing domain interaction predictions into subsets of low and high confidence. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.     

Coiled-Coil Formation on Lipid Bilayers—Implications for Docking and?Fusion Efficiency

Coiled-coil formation of four different oligopeptides was characterized in solution, on hydrogels, and on membranes by employing circular dichroism spectroscopy, surface plasmon resonance spectroscopy, attenuated total reflection infrared spectroscopy, and ellipsometry. Peptide sequences rich in either glutamic acid (E: E3Cys, i-E3Cys) or lysine (K: K3Cys, i-K3Cys) were used to represent minimal mimics of eukaryotic SNARE motifs. Half of the peptides were synthesized in reverse sequence, so that parallel and antiparallel heptad coiled-coil structures were formed. Either E-peptides or K-peptides were attached covalently to phospholipid anchors via maleimide chemistry, and served as receptors for the recognition of the corresponding binding partners added to solution. Attenuated total reflection infrared spectroscopy of single bilayers confirmed the formation of coiled-coil complexes at the membrane interface. Coiled-coil formation in solution, as compared with association at the membrane surface, displays considerably larger binding constants that are largely attributed to loss of translational entropy at the interface. Finally, the fusogenicity of the various coiled-coil motifs was explored, and the results provide clear evidence that hemifusion followed by full fusion requires a parallel orientation of α-helices, whereas antiparallel oriented coiled-coil motifs display only docking.     

TF — A novel cell-permeable and selective inhibitor of human protein kinase CK2 induces apoptosis in the prostate cancer cell line LNCaP

BackgroundAbnormally high activity of protein kinase CK2 is linked to various diseases including cancer. Therefore, the inhibition of CK2 is a promising therapeutic strategy to fight this disease.MethodsWe screened a library of synthetic molecules concerning their capacity to inhibit CK2. The activity of CK2 and their IC₅₀ and K_i values were determined by a capillary electrophoresis assay. The effects of the inhibitor in a cell culture model were analyzed by cell counting, a viability assay, cytofluorimetry and Western blot.ResultsThe best CK2 inhibitor found in this screen was 6,7-dichloro-1,4-dihydro-8-hydroxy-4-[(4-methylphenylamino)methylen]dibenzo [b,d]furan-3(2H)-one, which we refer to as “TF”. TF showed tight binding to CK2 with low IC₅₀ (29 nM) and K_i (15 nM) values. TF inhibited only seven out of 61 human kinases tested (> 70% inhibition). Incubation of LNCaP cells with 50 μM TF for 48 h decreased the intracellular CK2 activity by 50%, confirming that the inhibitor is membrane permeable. The decrease in activity was correlated with a severe reduction in cell viability. The reduction in cell viability is at least partly due to the induction of apoptosis.General significanceIn many cancers the protein kinase CK2 is significantly up-regulated and supports the neoplastic phenotype. New therapeutic strategies should be based on diverse reliable inhibitors to reverse the abnormal high levels to normal settings.     

Role of an N-Terminal Splice Segment in the Activation of the Cation Channel TRPM2 by ADP-Ribose and Hydrogen Peroxide

In the dysfunctional splice variant TRPM2-ΔN, a stretch of 20 amino acids (aa 537–556) is missing within the N-terminal cytosolic tail of the cation channel TRPM2. The ΔN-stretch overlaps with two IQ-like calmodulin-binding domains. Moreover, it contains two PxxP motifs implicated in protein–protein interactions. Here, we constructed variants to test whether any of these motifs may explain why TRPM2-ΔN does not respond to stimulation with either ADP ribose or hydrogen peroxide. Each of the two IQ-motifs could be removed without loss of channel function. Similarly, deletion of either one or both PxxP motifs had no effect. Moreover, the single point mutation D543E associated with bipolar disorder does not change the activation of TRPM2. We conclude that no functional role can be attributed to any of the structural motifs within the ΔN-stretch that may be a spacer segment for other functional sites in the N terminus.     

Phylogenetics of early branching eudicots: Comparing phylogenetic signal across plastid introns, spacers, and genes

Recent phylogenetic analyses revealed a grade with Ranunculales,Sabiales,Proteales,Trochodendrales,and Buxales as first branching eudicots,with the respective positions of Proteales and Sabiales still ...