François Rougeon

A procedure is described for the large-scale purification of light (L) and heavy (H) chain mRNAs from plasmacytomas produced in mice. Intact RNA is selectively precipitated in high yield from frozen tumors homogenized in 3 M LiCl and 6 M urea. L and H-chain mRNAs were purified by oligo(dT)-cellulose chromatography and either sucrose gradient centrifugation in conditions preventing aggregation or by means of high-resolution preparative gel electrophoresis under non-denaturing conditions. gamma 2a and alpha H-chain mRNAs sedimented as major components at 15.5 S and 16.5 S respectively, when L-chain mRNAs sedimented as 12-S species. H-chain mRNAs isolated by continuous elution during preparative gel electrophoresis were completely separated from both L-chain mRNA and residual 18-S rRNA, and migrated as single components of 1900 +/- 50 nucleotides on analytical denaturing gels. The partially purified H-chain mRNAs were translated into major components of molecular weights of 56,000 (gamma 2a) and 60,000 (alpha) in an mRNA-dependent rabbit reticulocyte lysate, whereas L-chain mRNAs yielded polypeptides of molecular weights of 25,000 (gamma) and 27,000 (chi). Up to 95% of the translation products directed by the purified mRNAs were immunoprecipitated using specific antisera. The purity of L and H-chain mRNAs was assessed by hybridization of corresponding cDNAs with excess recombinant plasmid DNA. The results indicated a minimum purity of 47% (gamma 2a), 62% (alpha), for H-chain mRNAs and 60% (chi), for L-chain mRNAs.

Rabies cDNA clones, obtained by "walking along the genome" using two successive DNA primers, have allowed the sequence determination of the genes encoding the N, M1, M2, G, and the beginning of the L protein as well as the rabies intergenic regions. Start and stop transcription signals located at the border of each gene encoding a protein have been identified and are similar to the corresponding signals from vesicular stomatitis virus (VSV) and Sendai virus. Except for limited stretches of the nucleoprotein, there is no homology between corresponding structural proteins of these three viruses. Rabies intergenic regions are variable both in length and sequence. Evidence for the existence of a remnant protein gene in the 423 nucleotide long G-L intergenic region is presented. This finding is discussed in terms of the evolution of unsegmented negative-strand RNA viruses.

Antibodies normally do not cross the blood‐brain barrier (BBB) and cannot bind an intracellular cerebral antigen. We demonstrate here for the first time that a new class of antibodies can cross the BBB without treatment. Camelids produce native homodimeric heavy‐chain antibodies, the paratope being composed of a single‐variable domain called VHH. Here, we used recombinant VHH directed against human glial fibrillary acidic protein (GFAP), a specific marker of astrocytes. Only basic VHHs ( e.g. , pI=9.4) were able to cross the BBB in vitro (7.8 vs. 0% for VHH with pI=7.7). By intracarotid and intravenous injections into live mice, we showed that these basic VHHs are able to cross the BBB in vivo , diffuse into the brain tissue, penetrate into astrocytes, and specifically label GFAP. To analyze their ability to be used as a specific transporter, we then expressed a recombinant fusion protein VHH‐green fluorescent protein (GFP). These “fluobodies” specifically labeled GFAP on murine brain sections, and a basic variant (pI=9.3) of the fusion protein VHH‐GFP was able to cross the BBB and to label astrocytes in vivo. The potential of VHHs as diagnostic or therapeutic agents in the central nervous system now deserves attention.—Li, T., Bourgeois, J.‐P., Celli, S., Glacial, F., Le Sourd, A.‐M., Mecheri, S., Weksler, B., Romero, I., Couraud, P.‐O., Rougeon, F., and Lafaye, P. Cell‐penetrating anti‐GFAP VHH and corresponding fluorescent fusion protein VHH‐GFP spontaneously cross the blood‐brain barrier and specifically recognize astrocytes: application to brain imaging. FASEB J. 26, 3969–3979 (2012). www.fasebj.org