Rabies cDNA clones, obtained by "walking This friding is discussed in terms of the evolution of unsegmented negative-strand RNA viruses.Unsegmented negative-strand RNA viruses are grouped into two families, Rhabdoviridae and Paramyxoviridae. Although they differ widely in their host range and their interaction with the host cell, all these viruses share a similar genomic organization and use the same multiplication strategy during their lytic cycle. The RNA genome is first used as template for the synthesis of monocistronic transcripts, then for replication into positive-strand genomes that are then replicated into the negative-strand genomes and these are encap-

The crystal structure of the catalytic core of murine terminal deoxynucleotidyltransferase (TdT) at 2.35 A Ê resolution reveals a typical DNA polymerase b-like fold locked in a closed form. In addition, the structures of two different binary complexes, one with an oligonucleotide primer and the other with an incoming ddATP-Co 2+ complex, show that the substrates and the two divalent ions in the catalytic site are positioned in TdT in a manner similar to that described for the human DNA polymerase b ternary complex, suggesting a common two metal ions mechanism of nucleotidyl transfer in these two proteins. The inability of TdT to accommodate a template strand can be explained by steric hindrance at the catalytic site caused by a long lariat-like loop, which is absent in DNA polymerase b. However, displacement of this discriminating loop would be suf®cient to unmask a number of evolutionarily conserved residues, which could then interact with a template DNA strand. The present structure can be used to model the recently discovered human polymerase m, with which it shares 43% sequence identity.

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). http://www.fasebj.org

Sialorphin is an exocrine and endocrine signaling mediator, which has been identified by a genomic approach. It is synthesized predominantly in the submandibular gland and prostate of adult rats in response to androgen steroids and is released locally and systemically in response to stress. We now demonstrate that the cell surface molecule to which sialorphin binds in vivo in the rat kidney is the membrane-anchored neutral endopeptidase (neprilysin; NEP, EC 3.4.24.11). NEP plays an important role in nervous and peripheral tissues, as it turns off several peptide-signaling events at the cell surface. We show that sialorphin prevents spinal and renal NEP from breaking down its two physiologically relevant substrates, substance P and Met-enkephalin in vitro. Sialorphin inhibited the breakdown of substance P with an IC50 of 0.4 -1 M and behaved as a competitive inhibitor. In vivo, i.v. sialorphin elicited potent antinociceptive responses in two behavioral rat models of injury-induced acute and tonic pain, the pin-pain test and formalin test. The analgesia induced by 100 -200 g͞kg doses of sialorphin required the activation of -and ␦-opioid receptors, consistent with the involvement of endogenous opioid receptors in enkephalinergic transmission. We conclude that sialorphin protects endogenous enkephalins released after nociceptive stimuli by inhibiting NEP in vivo. Sialorphin is a natural systemically active regulator of NEP activity. Furthermore, our study provides evidence that it is a physiological modulator of pain perception after injury and might be the progenitor of a new class of therapeutic molecules.

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Terminal deoxynucleotidyl transferase (TdT) catalyzes the condensation of deoxyribonucleotides on 3-hydroxyl ends of DNA strands in a template-independent manner and adds N-regions to gene segment junctions during V(D)J recombination. Although TdT is able to incorporate a few ribonucleotides in vitro, TdT discrimination between ribo-and deoxyribonucleotides has never been studied. We found that TdT shows only a minor preference for incorporation of deoxyribonucleotides over ribonucleotides on DNA strands. However, incorporation of ribonucleotides alone or in the presence of deoxyribonucleotides generally leads to premature chain termination, reflecting an impeded accommodation of ribo-or mixed ribo/deoxyribonucleic acid substrates by TdT. An essential catalytic aspartate in TdT was identified, which is a first step toward understanding the apparent lack of sugar discrimination by TdT.A traditional classification of polymerases, as DNA or RNA polymerases, relies on their ability to use dNTPs 1 or rNTPs as nucleotide substrates (1, 2). Sugar selectivity is extremely high, several orders of magnitude (3-7), allowing nucleic acid polymerases to accomplish specialized tasks in the presence of competing nucleotide substrates. Terminal deoxynucleotidyl transferase (TdT, EC2.7.7.31) is a DNA polymerase, which contributes to the diversification of antigen receptors by adding nucleotides, called N-regions, to gene segment junctions during V(D)J recombination (8,9). In vitro, TdT most efficiently polymerizes deoxynucleotides on 3Ј-hydroxyl ends of DNA strands in a template-independent manner (10, 11). However, shortly after the isolation of TdT, it was shown that TdT could add a few ribonucleotides to oligodeoxynucleotide initiators and incorporate ribonucleotides into growing DNA chains (12, 13). The methodology at that time allowed neither accurate determination of the reaction kinetic parameters nor calculation of the TdT selectivity factor for deoxyribonucleotides over ribonucleotides. Having recently developed a new method for producing large quantities of pure and unproteolyzed recombinant TdT in bacteria (14), we have been able to compare the activity of the two TdT isoforms found in the mouse (15), to undertake three-dimensional structural analyses (16), and to further improve the enzymatic characterization of the protein.Here, we present our analysis of the sugar selectivity of TdT, and our investigation of its enzymatic behavior in vitro in the presence of both ribo-and deoxyribonucleotides. We report that the short murine TdT, which is the isoform conserved across the vertebrate phylum (17), has only minor preference for the incorporation of deoxyribo-over ribonucleotides on DNA strands in vitro and that under conditions that reproduce the in vivo ribo/deoxyribonucleotide pool imbalance (18), incorporation of ribonucleotides by TdT leads to premature termination of chain elongation. As a first step toward analyzing the mechanisms underlying the apparent lack of sugar discrimination by TdT, unique among nucleic a...

Rearrangement of Ig H and L chain genes is highly regulated and takes place sequentially during B cell development. Several lines of evidence indicate that chromatin may modulate accessibility of the Ig loci for V(D)J recombination. In this study, we show that remodeling of V and J segment chromatin occurs before V(D)J recombination at the endogenous H and κ L chain loci. In recombination-activating gene-deficient pro-B cells, there is a reorganization of nucleosomal structure over the H chain JH cluster and increased DNase I sensitivity of VH and JH segments. The pro-B/pre-B cell transition is marked by a decrease in the DNase I sensitivity of VH segments and a reciprocal increase in the nuclease sensitivity of Vκ and Jκ segments. In contrast, JH segments remain DNase I sensitive, and their nucleosomal organization is maintained in μ+ recombination-activating gene-deficient pre-B cells. These results indicate that initiation of rearrangement is associated with changes in the chromatin structure of both V and J segments, whereas stopping recombination involves changes in only V segment chromatin. We further find an increase in histone H4 acetylation at both the H and κ L chain loci at the pro-B cell stage. Although histone H4 acetylation appears to be an early change associated with B cell commitment, acetylation alone is not sufficient to promote subsequent modifications in Ig chromatin.