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Nanoarchaeum equitansandIgnicoccus hospitalisrepresent a unique, intimate association of two archaea. Both form a stable coculture which is mandatory forN.equitansbut not for the hostI.hospitalis. Here, we investigated interactions and mutual influence between these microorganisms. Fermentation studies revealed that during exponential growth only about 25% ofI.hospitaliscells are occupied byN.equitanscells (one to three cells). The latter strongly proliferate in the stationary phase ofI.hospitalis, until 80 to 90% of theI.hospitaliscells carry around 10N.equitanscells. Furthermore, the expulsion of H2S, the major metabolic end product ofI.hospitalis, by strong gas stripping yields huge amounts of freeN.equitanscells.N.equitanshad no influence on the doubling times, final cell concentrations, and growth temperature, pH, or salt concentration ranges or optima ofI.hospitalis. However, isolation studies using optical tweezers revealed that infection withN.equitansinhibited the proliferation of individualI.hospitaliscells. This inhibition might be caused by deprivation of the host of cell components like amino acids, as demonstrated by13C-labeling studies. The strong dependence ofN.equitansonI.hospitaliswas affirmed by live-dead staining and electron microscopic analyses, which indicated a tight physiological and structural connection between the two microorganisms. No alternative hosts, including otherIgnicoccusspecies, were accepted byN.equitans. In summary, the data show a highly specialized association ofN.equitansandI.hospitaliswhich so far cannot be assigned to a classical symbiosis, commensalism, or parasitism.

The influence of the triterpenoid saponins 1-10 has been investigated on murine spleenocytes in the lymphocyte transformation test and on murine macrophages in an phagocytosis assay. The lymphocyte transformation test and the phagocytosis assay showed that the tested compounds have no stimulating effect. However, a significant inhibition of lymphocyte proliferation by the triterpenoid saponins 2, 6 and 10 was demonstrated.

l-Carrageenan is a polysulphated carbohydrate that antagonises some heparin-binding growth factors. We assessed the effect of l-carrageenan on the proliferation of a panel of cell lines, some of which require heparin-binding growth factors for mitogenesis. The importance of growth factor antagonism for the anti-proliferative activity was also determined. Cell proliferation was determined by cell counts and a tetrazolium dye (MTT) assay, and DNA synthesis was determined by thymidine incorporation. The proliferation of the basic fibroblast growth factor (bFGF)-dependent endothelial cell line FBHE was inhibited by daily administration of l-carrageenan in a dose-dependent manner [concentration inhibiting cell growth by 50% (IC50 value), approx. 0.5 microgram/ml]. However, excess bFGF did not reverse the inhibitory effect. DNA synthesis was completely inhibited by concentrations of l-carrageenan that nonetheless allowed significant protein synthesis to occur. The proliferation of the androgen-dependent prostate-carcinoma cell line LNCaP was also inhibited by l-carrageenan (IC50 value, 5.5 micrograms/ml) and the cells were arrested at the G1/S boundary. l-Carrageenan inhibited DNA synthesis in MCF-7 cells stimulated by bFGF and transforming growth factor alpha (TGF alpha) but not in those stimulated by insulin-like growth factor 1 (IGF-1). Blocking IGF-1-mediated DNA synthesis with anti-IGF-1 receptor antibody alpha IR3 enhanced the inhibitory activity of l-carrageenan against MCF-7 cells grown in serum. A number of other transformed and non-transformed cell lines were either partially inhibited or not inhibited by l-carrageenan. l-Carrageenan had low anti-coagulant activity. l-Carrageenan is a selective anti-proliferative agent and warrants further investigation for anti-angiogenic therapy (in view of its activity against endothelial cells) and for the treatment of androgen-dependent prostate cancer.

We have evaluated a series of laminarin sulphates with different degrees of sulphation (0.3-2.3) as antagonists of basic fibroblast growth factor (bFGF) and as inhibitors of the bFGF-dependent endothelial cell line FBHE. Inhibition of binding of bFGF by the laminarin sulphates increased with increasing degree of sulphation. Binding of bFGF to low affinity sites on BHK cells was inhibited more strongly than binding to high affinity sites. IC50 values for inhibition of binding to low and high affinity sites by the most highly sulphated laminarin sulphate (LAM S5; degree of sulphation 2.31) were 12 +/- 8 micrograms/ml and 69 +/- 66 micrograms/ml, respectively. LAM S5 dissociated bFGF from low affinity sites on BHK cells but not from high affinity sites. LAM S5 increased the electrophoretic mobility of bFGF indicating that LAM S5 binds directly to bFGF. LAM S5 reduced uptake of bFGF by FBHE cells by 67%. Increasing the degree of sulphation of laminarin sulphates increased the inhibition of bFGF-stimulated DNA synthesis of the endothelial cell line FBHE (IC50 for LAM S5 approx. 1 microgram/ml). There was no inhibition of DNA synthesis of FBHE cells by LAM S5 in the presence of 1 microgram/ml bFGF indicating that bFGF antagonism is involved in the anti-proliferative activity of this compound. LAM S5 may be of value against diseases associated with bFGF-dependent cell proliferation.

Cytokinesis requires the constriction of ESCRT-III filaments on the side of the midbody, where abscission occurs. After ESCRT recruitment at the midbody, it is not known how the ESCRT-III machinery localizes to the abscission site. To reveal actors involved in abscission, we obtained the proteome of intact, post-abscission midbodies (Flemmingsome) and identified 489 proteins enriched in this organelle. Among these proteins, we further characterized a plasma membrane-to-ESCRT module composed of the transmembrane proteoglycan syndecan-4, ALIX and syntenin, a protein that bridges ESCRT-III/ALIX to syndecans. The three proteins are highly recruited first at the midbody then at the abscission site, and their depletion delays abscission. Mechanistically, direct interactions between ALIX, syntenin and syndecan-4 are essential for proper enrichment of the ESCRT-III machinery at the abscission site, but not at the midbody. We propose that the ESCRT-III machinery must be physically coupled to a membrane protein at the cytokinetic abscission site for efficient scission, uncovering common requirements in cytokinesis, exosome formation and HIV budding., ESCRT filaments drive the final abscission between two daughter cells but how they physically interact with the membrane is unclear. Using proteomics, the authors show that syndecan-4/syntenin/ALIX couples the ESCRT machinery to the abscission site and thus promotes efficient abscission.