The D6 heptahelical membrane protein, expressed by lymphatic endothelial cells, is able to bind with high affinity to multiple proinflammatory CC chemokines. However, this binding does not allow D6 to couple to the signaling pathways activated by typical chemokine receptors such as CC-chemokine receptor-5 (CCR5). Here, we show that D6, like CCR5, can rapidly internalize chemokines. However, D6-internalized chemokines are more effectively retained intracellularly because they more readily dissociate from the receptor during vesicle acidification. These chemokines are then degraded while the receptor recycles to the cell surface. Interestingly, D6-mediated chemokine internalization occurs without bringing about a reduction in cell surface D6 levels. This is possible because unlike CCR5, D6 is predominantly localized in recycling endosomes capable of trafficking to and from the cell surface in the absence of ligand. When chemokine is present, it can enter the cells associated with D6 already destined for internalization. By this mechanism, D6 can target chemokines for degradation without the necessity for cell signaling, and without desensitizing the cell to subsequent chemokine exposure.

The vapor pressures above Mo03, M04O11, Mo02, W03 and W02 have been measured by the Knudsen effusion method. MoOs(s) vaporizes to (Mo03)3(g) while Mo40u(s) disproportionates to (Mo03)3(g) and Mo02(s), the heats of vaporization of Mo03(s) and of decomposition of MoiOu(s) being AHm = 79.7 kcal./mole of (Mo03)3 and AH mo -68.7 kcal./mole of (Mo03)3, respectively. Some Mo02(s) vaporizes to Mo02(g) with AHq = 129.8 kcal./mole, but most of the Mo02(s) disproportionates to Mo03(g) and Mo(s) with AH'¿ = 133.6 kcal./mole of Mo03. WOi(s) vaporizes to (W03)3(g) with AH mo = 108.0 kcal./mole of (W03)3 and W02(s) disproportionates to (W03)3(g) and W(s) with / = 90.2 kcal./mole of (W03)3. The calculated heat of dissociation for Mo02(g) is Do = 284.6 kcal./mole and for MoOs(g) is D°-410.3 kcal./mole. The heat of decomposition of (Mo03)3(g) to the monomer Mo03(g) is AH = 220.9 kcal./mole of the trimer. This research is concerned with the investigation of the vapor or dissociation pressures of M0O3, M0O2, W03, W03, and the stability of the intermediate compounds. There has been substantial disagreement among molybdenum trioxide vapor pressure data obtained by the Knudsen effusion method,3 by transpiration measurements,4 and by the rate of evaporation method.5 W03 vapor pressures have been investigated by Ueno.3 Calculations using Kelley's6 and Brewer's7 data show that M0O2 and W02 should decompose to the metal and the trioxide. No measurements are available on the lower oxides of either metal. Apparatus and Experimental ProcedureThe heating was done entirely by induction, using a General Electric or a Westinghouse 20 kilowatt electronic heater.Three furnaces were used for the research. A furnace for temperatures below the optical range was used for vapor pressure measurements on molybdenum trioxide and molybdenum dioxide-molybdenum trioxide mixtures. The remaining measurements were made in either the high temperature Pyrex glass furnace or the metal furnace previously described.8 The only change made on the high (1) This work was supported in part by the Office of Naval Research under contract with the Ohio State University Research Foundation.

There is much interest in chemokine receptors as therapeutic targets in diseases such as AIDS, autoimmune and inflammatory disorders, and cancer. Hampering such studies is the lack of accurate three-dimensional structural models of these molecules. The CC-chemokine receptor D6 is expressed at exceptionally high levels in heterologous transfectants. Here we report the purification and biochemical characterization of milligram quantities of D6 protein from relatively small cultures of transfected mammalian cells. Importantly, purified D6 retains full functional activity, shown by displaceable binding of 125I-labelled MIP-1beta (macrophage inflammatory protein-1beta) and by complete binding of the receptor to a MIP-1alpha affinity column. In addition, we show that D6 is decorated on the N-terminus by N-linked glycosylation. Mutational analysis reveals that this glycosylation is dispensable for ligand binding and high expression in transfected cells. Metabolic labelling has revealed the receptor to also be sulphated and phosphorylated. Phosphorylation is ligand independent and is not enhanced by ligand binding and internalization, suggesting similarities with the viral chemokine receptor homologue US28. Like US28, an analysis of the full cellular complement of D6 in transfected cells indicates that >80% is found associated with intracellular vesicular structures. This may account for the high quantities of D6 that can be synthesized in these cells. These unusual properties of D6, and the biochemical characterization described here, leads the way towards work aimed at generating the three-dimensional structure of this seven-transmembrane-spanning receptor.

We report the creation of permanent 3D configurations of cells, at predefined positions, within a gelatin matrix. The technique used holographic optical tweezers to manipulate individual E. coli within a solution comprising monomer precursors. The matrix was then set and after the laser beam was removed, we were able to demonstrate that the structures remained intact for many days. We were also able to demonstrate that, in the presence of appropriate nutrients, the E. coli survived within the gelatin matrix for several days. The technique could have a number of potential future applications, including the arrangement of a variety of different cell types in complex architectures, as motifs for promoting tissue differentiation and growth within the field of cell engineering.