The need to develop a blood substitute is now urgent because of the increasing concern over blood-transmitted viral and bacterial pathogens. Cell-free haemoglobin solutions and human haemoglobin synthesized in Escherichia coli and Saccharomyces cerevisiae have been investigated as potential oxygen-carrying substitutes for red blood cells. But these haemoglobins cannot be used as a blood substitute because (1) the oxygen affinity in the absence of 2,3-bisphosphoglycerate is too high to allow unloading of enough oxygen in the tissues, and (2) they dissociate into alpha beta dimers that are cleared rapidly by renal filtration, which can result in long-term kidney damage. We have produced a human haemoglobin using an expression vector containing one gene encoding a mutant beta-globin with decreased oxygen affinity and one duplicated, tandemly fused alpha-globin gene. Fusion of the two alpha-globin subunits increases the half-life of this haemoglobin molecule in vivo by preventing its dissociation into alpha beta dimers and therefore also eliminates renal toxicity.

Thrombospondin, a high molecular weight glycoprotein secreted by platelets in response to activation by thrombin, has been identified by immunofluorescence in bovine aortic endothelial cells, human foreskin fibroblasts, and human aortic smooth muscle cells. Immunofluorescence patterns were found to be similar using antisera raised to thrombospondins purified either from bovine aortic endothelial cells or from human platelets. Radioimmune precipitation of pulse-labeled cellular proteins confirmed the presence of thrombospondin in positively stained cells. A sensitive quantitative enzyme-linked immunosorbent assay (ELISA) was developed and used to determine that the accumulation of secreted thrombospondin was similar for endothelial cells and fibroblasts but was higher for smooth muscle cells. The presence of thrombospondin in a variety of cells suggests that its function may not be limited to an involvement in platelet interactions.Thrombospondin (TS), a high molecular weight glycoprotein, is released from a-granules after activation of platelets by thrombin (1, 2). After release, the protein binds to the activated platelet surface in a calcium-dependent fashion (3) and may participate in platelet-platelet interactions (4). Earlier work in this laboratory (5) identified a high molecular weight glycoprotein secreted by endothelial cells in culture that represented a substantial portion of the noncollagenous protein synthesized and secreted by these cells. This glycoprotein was subsequently shown to be indistinguishable from TS by a variety of criteria, including co-purification, molecular weight, amino acid composition, immunological cross-reactivity, and peptide maps (6).Although TS has been shown to have lectinlike activity in platelet-platelet interactions (4), its function in endothelial cells is not known. This led us to examine other cells in culture for the synthesis and secretion of TS. We have now identified TS in a variety of mesenchymal cells. Since TS is secreted and deposited in the cell layer of these cells in culture, we postulate that it may function as a matrix protein in vivo. MATERIALS AND METHODS Cell CultureBovine aortic endothelial (BAE) ceils were provided by Dr. S. Schwartz (University of Washington). They were maintained in Waymouth's medium supplemented with 10% fetal calf serum (FCS) (Reheis Chemical Company, Phoenix, AZ), penicillin (100 U/nil), and streptomycin (100 gg/ml). Human dermal fibroblasts were isolated from foreskin explants and maintained in Dulbecco's modified Eagle's medium (DME) supplemented with 10% FCS and antibiotics. Smooth muscle ceils were obtained from human aortae and were provided by Dr. Russell Ross (University of Washington). They were maintained in DME supplemented with 20% FCS and antibiotics. Ceils were grown at 37°C in an atmosphere of 5% CO2. Purification of Thrombospondins BAE cell TS was isolated according to the method of McPherson el al. (6).Human platelet TS was isolated from l-d-old blood bank platelets according to the procedure of Lawler a...

Thrombospondin (TS), a 450,000 molecular weight glycoprotein, is released from alpha-granules of thrombin-activated platelets and is secreted and incorporated into the extracellular matrix by several cell types in culture. We have examined the effects of cell density and transformation on the production of TS in cell culture. The levels of TS, per cell, in the culture medium of endothelial cells, smooth muscle cells, and fibroblasts were greater at lower cell densities; in fibroblasts the levels of two other extracellular matrix proteins, fibronectin and collagen, were unaffected by cell density. Our evidence indicates that the higher levels of TS in the culture medium, determined for lower-density cells, were achieved by an increased secretion of the protein rather than by a reduction in degradation or incorporation into the extracellular matrix. TS production by normal and transformed WI-38 fibroblasts was the same, although the fibronectin level in the culture medium of the transformed cells was substantially decreased. These findings suggest that the production of TS by cells in culture is regulated in a different fashion from that of fibronectin or collagen.

We produced human monoclonal antibody that demonstrated specific reactivity to the K1 capsule of Escherichia coli and the group B polysaccharide of Neisseria meningitidis. The antibody was nonreactive with several strains of K1- E. coli and other gram-negative bacteria. All E. coli K1 clinical isolates tested were reactive with the antibody. When assayed for in vitro opsonophagocytic ability, the antibody caused bacterial removal only in the presence of human complement and neutrophils, an observation suggesting a non-bacteriolytic, neutrophil-dependent killing mechanism. Finally, and perhaps most importantly, the antibody was highly protective for infectious disease when used prophylactically in three animal models. The data suggest a potential use for human monoclonal antibodies in preventing and/or treating infections of the blood.

Recombinant human hemoglobin rHbl. I has been genetically engineered with the replacement of the wild-type valine residues at all N-termini with methionine, an Asn 108 Lys substitution on the beta globins, and a fusion of the two alpha globins with a glycine linker. When rHbl.1 was expressed in Escherichia coli, methylation of the N-terminal methionine of the alpha globin was discovered. Another mutant has been engineered with the alpha globin gene coding for N-terminal methionine followed by an insertion of alanine. Characterization of expressed hemoglobin from this variant revealed a methylated N-terminal alanine that occurred through two posttranslational events: initial excision of the N-terminal methionine, followed by methylation of alanine as the newly generated N-terminus. No methylation was observed for variants expressed with wild-type valine at the N-terminus of the alpha globin. The methylation of N-terminal amino acids was attributed to a specific protein sequence that can trigger methylation of proteins expressed in E. coli. Here we demonstrate that proline at position 4 in the protein sequence of alpha globin seems an essential part of that signaling. Although N-terminal methylation has been observed previously for native E. coli proteins with similar N-terminal sequences, methylation of the recombinant globins has allowed further delineation of the recognition sequence, and indicates that methylation of heterologous proteins can occur in E. coli.