Membrane glycolipid and phospholipid composition of lipopolysaccharide-responsive and -nonresponsive murine B lymphocytes

Neutral glycolipids, gangliosides, and phospholipids present on membranes of unstimulated or lipopolysaccharide (LPS)-stimulated B cells were analyzed in LPS-responsive C3H/HePAS and LPS-nonresponsive C3H/HeJ mice. In the set of neutral glycolipids, asialo GM1 reacted preferentially with galactose oxidase but was not detectable with monospecific antibodies during immunocytofluorescence analysis. Another, more polar, neutral glycolipid appeared exclusively after stimulation of responsive B cells. Among the membrane gangliosides 1 to 5 that were able to react with galactose oxidase on B cells, ganglioside 3 was not detected in the mutant strain, and its absence was counterbalanced by the presence of a larger amount of ganglioside 1. The biosynthesis of total membrane phospholipids and the balance between phosphatidylethanolamine and phosphatidylcholine were significantly different in the two mouse strains examined and were quantitatively and qualitatively modified during the mitogenic response to LPS.</jats:p

Antibodies raised against a peptide corresponding to a Gs alpha domain reveal a vimentin-associated protein.

In an attempt to localize the guanine nucleotide binding protein Gs alpha by immunocytochemistry, we synthetized peptides corresponding to several stretches of residues deduced from the published cDNA sequence of Gs alpha and raised antibodies against them. Among the peptides, one corresponding to residues 367-381 elicited antibodies that immunocytochemically detected, at the optical level, what appeared to be vimentin in several cells and tissues. Studies at the ultrastructural level confirmed this observation and also showed weak staining of some areas of plasma membranes of glial and nerve cells. Analysis by Western blots of rat brain subcellular fractions indicated that: (a) the protein stained by the anti-peptide antibodies was associated with the cytoskeleton; and (b) it was not vimentin but a protein of higher molecular weight, 65 KD. We accordingly suggest that the Gs alpha-derived peptide elicited two types of antibodies, one recognizing Gs alpha in fixed tissues, the other recognizing an epitope located in a vimentin-associated protein. This study emphasizes the caution that is needed when conclusions are drawn on the basis of immunocytochemical studies using antipeptide antibodies. </jats:p

Plasmidic versus Insertional Cloning of Heterologous Genes in Mycobacterium bovis BCG: Impact on In Vivo Antigen Persistence and Immune Responses

Bivalent recombinant strains of Mycobacterium bovis BCG (rBCG) expressing the early regulatory nef and the structural gag(p26) genes from the simian immunodeficiency virus (SIV) SIVmac251 were engineered so that both genes were cotranscribed from a synthetic operon. The expression cassette was cloned into a multicopy-replicating vector, and the expression levels of both nef and gag in the bivalent rBCG(nef-gag) strain were found to be comparable to those of monovalent rBCG(nef) or rBCG(gag) strains. However, extrachromosomal cloning of the nef-gag operon into a replicative plasmid resulted in strains of low genetic stability that rapidly lost the plasmid in vivo. Thus, the nef-gag operon was inserted site specifically into the BCG chromosome by means of mycobacteriophage Ms6-derived vectors. The resulting integrative rBCG(nef-gag) strains showed very high genetic stability both in vitro and in vivo. The in vivo expression of the heterologous genes was much longer lived when the expression cassette was inserted into the BCG chromosome. In one of the strains obtained, integrative cloning did not reduce the expression levels of the genes even though a single copy was present. Accordingly, this strain induced cellular immune responses of the same magnitude as that of the replicative rBCG strain containing several copies of the genes