Richard Chaby

Colorimetric estimation of 3-deoxy-d-manno-octulosonic acid in oligosaccharides with diphenylamine

Abstract A method for the colorimetric estimation of 3-deoxy- d -manno-octulosonic acid with diphenylamine is described. The aldulosonic acid can be estimated in the presence of a tenfold excess of other sugars.

Specific and cross-reacting monoclonal antibodies to Bordetella parapertussis and Bordetella bronchiseptica lipopolysaccharides.

Three groups of monoclonal antibodies (mAbs) were produced that would be useful for immunochemical typing and diagnosis of infections due to Bordetella species, and for the structural analysis of their lipopolysaccharides. PP6, a representative of the first group, recognizes an epitope shared by smooth-type Bordetella parapertussis and Bordetella bronchiseptica lipopolysaccharides (LPS). This epitope is carried by structurally identical polymeric O-chains (POC) present on both LPS molecules. PP8 and PP9 are representatives of the second group of mAbs. The interaction of PP8 and PP9 with B. parapertussis and B. bronchiseptica LPS requires POC, but periodate-sensitive sugar units of the core are also involved in the binding. The mAb BRg1 belongs to the third group, and specifically recognizes B. bronchiseptica LPS. Binding and inhibition studies with various Bordetella LPS molecules, and with their polysaccharide fragments, indicated that BRg1 interacts with a structure located at the hinge between the POC and a core region of the B. bronchiseptica LPS containing periodate-resistant sugars. This suggests that the structures of the hinge regions of the B. parapertussis and B. bronchiseptica LPS are different.

Synthetic peptides representing the N-terminal segment of surfactant protein C modulate LPS-stimulated TNF-α production by macrophages

Surfactant protein C (SP-C) consists of a hydrophobic α-helix inserted in pulmonary surfactant membranes, and a more polar N-terminal palmitoylated segment exposed to the aqueous phase. Previously, we showed that SP-C inserted in lipid vesicles interacts with bacterial lipopolysaccharide (LPS) and reduces LPS-elicited responses. As the N-terminal segment of SP-C was the most likely region responsible for these effects, a set of synthetic analogs of this stretch (SPC(1-13) ) were studied. Binding studies showed that SPC(1-13) binds LPS to the same extent as porcine SP-C under lipid-free conditions. In the absence of serum, both, palmitoylated and non-palmitoylated analogs enhanced the binding of tritiated LPS to macrophages as well as the LPS-induced production of TNF-α by these cells. These effects were reversed in the presence of serum; the analogs reduced the production of TNF-α in LPS-stimulated macrophages, probably by interfering with the formation of LPS/CD14/LBP complexes as suggested by analysis of the fluorescence emitted by a FITC derivative of Re-LPS. Our data indicate that water-soluble analogs of the N-terminal segment of SP-C can reduce LPS effects in the presence of serum, and thus might help in the design of new derivatives to fight endotoxic shock and pro-inflammatory events.

Colorimetric estimation of ethanolamine with p-benzoquinone in the nanomole range.

Abstract Ethanolamine reacts with p -benzoquinone in alkaline medium to give a product of underermined structure, the absorption of which at 348 nm can be used for the estimation of the base. Conditions are described in which the interference of O -phosphorylethanolamine, glucosamine, and neutral sugars is eliminated. The method was used to estimate the ethanolamine content of phospholipids alone and admixed to interfering substances. It is unreliable in the presence of amino acids or proteins.

The Splenic Pool of Mouse Stem Cells: in vitro Differentiation and Expression of the BP-1 Alloantigen on Cells of the Lymphoid and Myeloid Lineages

The relative paucity of data about the development of the stem cell pool present in the spleen prompted this study. During in vitro cultures of B-enriched lymphocytes from mouse spleens and in the presence of a culture supernatant of WEHI-3 cells (WEHI-SUP), a population of cells expressing the BP-1 antigen appears progressively, reaches an optimal size 8 days after initiation of the culture, and disappears on day 28. In 8-day-old cultures, a minor population of cells bearing both BP-1 and B220 can be detected. The growth of this cell population, with characteristics of the B lymphoid lineage (pro-B), is strictly dependent on the presence of WEHI-SUP in the medium. After 2 weeks of culture, the BP-1 antigen is expressed on a cell population, which is essentially constituted of B220-, polynuclear cells. The BP-1 antigen, which is considered as characteristic of early cells of the B lymphoid lineage, can therefore also be expressed on cells of the myeloid lineage. The injection of BP-1+ or B220+ cells in irradiated mice can hardly reconstitute their B cell pool, whereas BP-1- and B220- cells are much more efficient in vivo progenitors of this cell lineage.