Claude Benicourt

Distinct neuroprotective profiles for σ ligands against N-methyl-d-aspartate (NMDA), and hypoxia-mediated neurotoxicity in neuronal culture toxicity studies

Substantiating evidence has raised the possibility that sigma ligands may have therapeutic potential as neuroprotective agents in brain ischemia. It has been suggested that the neuroprotective capacity of sigma ligands is related primarily to their affinity for the NMDA receptor complex and not to any selective action at the sigma binding site. However, sigma specific ligands, devoid of significant affinity for the NMDA receptor, are also neuroprotective via an inhibition of the ischemic-induced presynaptic release of excitotoxic amino acids. In the present study, we have investigated the potential neuroprotective effect of a comprehensive series of sigma ligands, with either significant (sigma/PCP) or negligible (sigma) affinity for the PCP site of the NMDA receptor, in order to delineate a selective sigma site-dependent neuroprotective effect. For this aim, we have employed two different neuronal culture toxicity paradigms implicating either postsynaptic-mediated neurotoxicity, (brief exposure of cultures to a low concentration of NMDA or Kainate) or pre- and postsynaptic mechanisms (exposure to hypoxic/hypoglycemic conditions). Only sigma ligands with affinity for the NMDA receptor [(+) and (-) cyclazocine, (+) pentazocine, (+) SKF-10047, ifenprodil and haloperidol] were capable of attenuating NMDA-induced toxicity whereas the sigma [(+)BMY-14802, DTG, JO1784, JO1783, and (+)3-PPP] and kappa-opioid [CI-977, U-50488H] ligands, with very low affinity for the NMDA receptor, were inactive. The rank order of potency, based on the 50% protective concentration (PC50) value, of sigma/PCP ligands against NMDA-mediated neurotoxicity correlates with their affinity for the PCP site of the NMDA receptor, and not with their affinity for the sigma site. In addition sigma/PCP, sigma or kappa-opioid ligands failed to attenuate kainate-mediated neurotoxicity. On the other hand, sigma/PCP, sigma and kappa-opioid ligands were potent inhibitors of hypoxia/hypoglycemia-induced neurotoxicity, although their neuroprotective potency did not correlate with their affinity for either the sigma or PCP binding sites. In conclusion, the ability of sigma and kappa-opioid ligands to attenuate hypoxia/hypoglycemia, but not NMDA or kainate-induced toxicity, suggests that these drugs exert their neuroprotective role by a predominantly presynaptic mechanism possibly by inhibiting ischemic-mediated glutamate release.

Translation of TYMV RNA into high molecular weight proteins

The wheat germ cell-free system leads to the translation of TYMV RNA into mature coat protein and several other polypeptides [l] , although not to a polypeptide whose size corresponds to the full translation of the viral genome. TYMV contains not only complete viral genomic RNA (2 X lo6 daltons), but also several other classes of RNA molecules ranging from 2-0.25 X 10” daltons [2,3] . The complete viral genome by itself is infectious [2], and therefore contains all the information necessary to generate new virus particles; in a wheat germ cell-free system, however, it directs the synthesis of only small amounts of coat protein molecules, if at all, thereby suggesting that the coat protein gene is masked in such a system. In order to determine whether TYMV RNA can direct the synthesis of high molecular weights proteins (>lOO 000 daltons), we turned to the use of the mRNA-dependent reticulocyte lysate which has been shown by Pelham and Jackson [4] to translate TMV RNA into one protein of 130 000 daltons. Our results show that in the reticulocyte system total TYMV RNA is efficiently translated into two high molecular weight proteins, one of 195 000 daltons, the other of 150 000 daltons, as well as into coat protein (20 000 daltons). The largest protein is

Differential translation of turnip yellow mosaic virus mRNAs in vitro.

Abstract Total TYMV RNA was incubated in a reticulocyte lysate, and the initiation peptides of the main proteins synthesized in vitro (195 K, 150 K and 20 K daltons) analyzed after tryptic digestion. The 195 K and the 150 K dalton proteins present analogous patterns, different from the one obtained with the 20 K dalton protein (coat protein), suggesting that only one initiation site exists on the genomic RNA for the synthesis of the two high molecular proteins. The results of competition experiments between genomic and coat protein mRNA indicate that the ribosomes have a much greater affinity for the coat protein mRNA. This may represent a regulatory mechanism for the preferential amplification of coat protein synthesis in the infected cells.

Tryptic cleavage of gastric lipases: location of the single disulfide bridge.

Human (HGL) and rabbit (RGL) gastric lipases were cleaved by trypsin and the resulting peptides were characterized. Exposure of HGL to trypsin led to the production of three identified fragments (H1, H2 and H3) resulting from cleavage sites at Lys-4 and Arg-229. Fragments H2 (Lys-4-Arg-229) and H3 (Glu-230-Lys-379) were derived from fragment H1 (Lys-4-Lys-379). The single disulfide bridge (Cys-236-Cys-244) of the molecule is localized in fragment H3. Out of the three cysteine residues conserved in all known gastric lipases, the free sulfhydryl group (Cys-227) was localized in fragment H2. Immunoblots, carried out with the tryptic fragments of HGL and anti-HGL mAbs, revealed that five inhibitory mAbs immunoreacted selectively with the N-terminal fragment H2, whereas two other non inhibitory mAbs immunoreacted exclusively with the C-terminal fragment H3. Trypsin also cleaved RGL at two sites (Arg-55 and Arg-229) leading to four identifiable fragments (R1, R2, R3 and R4). One cleavage site (Arg-229) was found to be identical in both RGL and HGL. We propose that this latter site is localized between the two domains of native gastric lipases.

Cell free translation of mRNA coding for human and murine calcitonin

Calcitonin (CT), the hypocalcemic hypophosphatemic hormone, is secreted by the thyroidal C-cells in mammals and by the ultimobranchial gland in submammalian species. We have reported the presence of several immunoreactive forms [l-3] larger than calcitonin monomer in human medullary carcinoma of the thyroid (MCT), a differentiated calcitonin secreting tumour [4]. These forms could represent higher molecular weight biosynthetic precursors of human CT. So far studies on the biosynthesis of calcitonin in 3 animal species have provided evidence for the presence of a prohormone of Mr 7000 (trout) [S], 13 500 (chicken) [6] and 11 800 (human) [7]. Using mRNA extracted from MCT and a murine transmissible tumour, primary translation product was reported to be a molecule of Mr 65 000 [8]. In [9] M, 15 000 was reported for the primary translation product of mRNA extracted from codfish or from a murine transmissible tumour, while in [lo] Mr 15 000 also for the primary translation product of mRNA extracted from MCT was reported. We report here studies on the primary tran~ation product of mRNA extracted from MCT. As work concerning mammalian mRNA was done with tumoral tissue, we have also extracted and translated mRNA from a normal tissue (rat thyroid) as in this species a considerable sequence homology with human CT (hCT) exists [ 111.

Recognition of TMV RNA by the tRNA nucleotidyltransferase

Within the last few years, evidence has accumulated that several plant viral RNA’s possess a tRNA-like structure at their 3’ end, since they can be charged with an amino acid, and since they are recognized by a series of enzymes specific of tRNAs [l-9]. Thus, the RNA of Turnip Yellow Mosaic Virus (TYMV) and of two other tymoviruses can be aminoacylated with valine [ 1,2,7], and the RNA of the members of the bromovirus group and that of Cucumber Mosaic Virus by tyrosine (8 and R. J. Kohl and T. C. Hall, personal communication). It has been reported that TMV RNA can accept histidine [4,9], and moreover that the histidyl-RNA can form a ternary complex with wheat embryo elongation factor EF-1 and GTP [4]. Further, the sequence of the pentanucleotide at the 3’ end of the viral RNA of 2 X lo6 daltons [lo] is known to be -GCCCA [ 111. It is interesting to note that only few tRNAs contain a cytidine residue in the fourth position from the 3’ end [ 121. Those which are known are acceptor only of histidine or of proline. Because of the tRNA-like properties of this viral RNA, and since it contains the 3’ terminal sequence --CCA common to all tRNAs, we have investigated whether it could also be a substrate of the tRNA nucleotidyltransferase (CCA-enzyme), the enzyme capable of adding back CCA to tRNA deprived of this 3’ terminal sequence. Our conclusion is that under conditions wherein the CCA-enzyme ‘repairs’ tRNAs, it also adequately ‘repairs’ TMV RNA, adding back the -CCA sequence to the RNA from which the 3’ terminal trinucleotide has been previously rem&d. As this work was