In development, tissue regeneration or certain diseases, angiogenic growth leads to the expansion of blood vessels and the lymphatic vasculature. This involves endothelial cell proliferation as well as angiogenic sprouting, in which a subset of cells, termed tip cells, acquires motile, invasive behaviour and extends filopodial protrusions. Although it is already appreciated that angiogenesis is triggered by tissue-derived signals, such as vascular endothelial growth factor (VEGF) family growth factors, the resulting signalling processes in endothelial cells are only partly understood. Here we show with genetic experiments in mouse and zebrafish that ephrin-B2, a transmembrane ligand for Eph receptor tyrosine kinases, promotes sprouting behaviour and motility in the angiogenic endothelium. We link this pro-angiogenic function to a crucial role of ephrin-B2 in the VEGF signalling pathway, which we have studied in detail for VEGFR3, the receptor for VEGF-C. In the absence of ephrin-B2, the internalization of VEGFR3 in cultured cells and mutant mice is defective, which compromises downstream signal transduction by the small GTPase Rac1, Akt and the mitogen-activated protein kinase Erk. Our results show that full VEGFR3 signalling is coupled to receptor internalization. Ephrin-B2 is a key regulator of this process and thereby controls angiogenic and lymphangiogenic growth.

This paper reports the generation of a self-assembled monolayer (SAM) that selectively binds proteins whose primary sequence terminates with a His-tag: a stretch of six histidines commonly incorporated in recombinant proteins to simplify purification. The SAM was prepared by the adsorption onto a gold surface of a mixture of two alkanethiols: one thiol that terminated with a nitrilotriacetic acid (NTA) group, a group that forms a tetravalent chelate with Ni(II), and a second thiol that terminated with a tri(ethylene glycol) group, a group that resists protein adsorption. His-tagged proteins bound to the SAM by interaction of the histidines with the two vacant sites on Ni(II) ions chelated to the surface NTA groups. Studies with model proteins showed the binding was specific for His-tagged proteins and required the presence of Ni(II) on the surface. Immobilized His-tagged proteins were kinetically stable in buffered saline at pH 7.2 but could be desorbed by treatment with 200 mM imidazole. Surface plasmon resonance studies for two model systems showed that His-tagged proteins adsorbed on the NTA-SAM retained a greater ability to participate in binding interactions with proteins in solution than protein immobilized in a thin dextran gel layer by covalent coupling.

In yeast strains bearing the point mutation called GAL11P (for potentiator), certain GAL4 derivatives lacking any classical activating region work as strong activators. The P mutation confers upon GAL11, a component of the RNA polymerase II holoenzyme, the ability to interact with a portion of the dimerization region of GAL4. The region of GAL11 affected by the P mutation is evidently functionally inert in ordinary cells, suggesting that this mutation is of no functional significance beyond creating an artificial target for the GAL4 dimerization fragment. From these observations and further analyses of GAL11, we propose that a single activator-holoenzyme contact can trigger gene activation simply by recruiting the latter to DNA.

No abstract

A novel B-cell-specific transcription factor, BSAP, was identified as a mammalian homolog of the sea urchin protein TSAP, which interacts with the promoters of four tissue-specific late histone H2A-2 and H2B-2 genes. As shown by mobility-shift, methylation interference, and mutational analyses, the mammalian protein BSAP recognizes all four sea urchin binding sites in a manner indistinguishable from TSAP; however, the two proteins differ in molecular weight. BSAP is exclusively restricted to the B-cell lineage of lymphoid differentiation. Its expression appears to be activated during pro-B-cell development, is abundant at the pre-Band mature B-cell stages, but is absent in terminally differentiated plasma cells. Moreover, BSAP is clearly a Bcell-specific transcription factor, as a wild-type but not a mutant TSAP-binding site of the sea urchin functions only in transfected B cells as an upstream promoter element. Competition experiments did not reveal any high-affinity binding site for BSAP in known regulatory regions of immunoglobulin and class II major histocompatibility (MHC) genes, suggesting that BSAP is a regulator of a different set of B-lymphoid-specific genes.