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During the Fmoc solid-phase synthesis of reduced peptide bond analogues, we observed that the trityl protection of an asparagine residue in the vicinity of a reduced peptide bond is not cleaved completely after the final trifluoroacetic acid deprotection step. The relative position of the Asn side-chain amine and of the aminomethylene bond as well as the preferential protonation of the secondary amine can be used to explain this phenomenon. We show that longer deprotection times or the use of methyl-trityl protection partially improves the yield of the Asn-deprotected peptide whereas xanthenyl protection totally overcomes this problem.

The human HIV transactivator protein Tat is essential for efficient viral transcription that occurs by a complex mechanism involving interaction of Tat with the TAR RNA element. This interaction appears to require the mediation of a cellular protein, cyclin T1. However, the possibility that Tat and TAR associate in a binary Tat-TAR complex has been little investigated. Using a chemically synthesized active Tat protein, the kinetic and equilibrium parameters of its interaction with TAR were determined by surface plasmon resonance technology. Independently of partner and method of immobilization onto the sensor chip, the association (k(a) = 5-9 x 10(5) M(-1) s(-1)) and dissociation rate constants (k(d) = 1.7-4.3 x 10(-3) s(-1)) yielded similar equilibrium dissociation constants (K(d) = 2-8 nM). A truncated peptide encompassing residues 30-86 of Tat did not bind to TAR at all. We conclude that Tat can form a high-affinity complex with TAR in the absence of cyclin T1 and that the N-terminal domain of Tat is essential for this interaction, suggesting a conformational link between this domain and the basic domain of Tat. These results are important in our quest for developing therapeutic compounds that impair viral replication.

Grenoble Test Source (GTS) is a room temperature electron cyclotron resonance ion source whose purpose is to deepen the knowledge of this type of device. GTS was designed according to magnetic scaling laws determined with the SERSE source [Hitz et al., Rev. Sci. Instrum. 73, 509 (2002); Gammino et al., ibid. 72, 4090 (2001)] while keeping enough flexibility in terms of magnetic confinement and rf heating to determine best conditions for the production of intense beams of any charge state. First results were presented 1 year ago [Hitz et al., 8th European Particle Accelerator Conference, 2002; 15th International Workshop on ECR Ion Sources, 2002]. Since then, some improvements have been performed mostly in the magnetic confinement, beam extraction and analysis. Updated ion beam intensities are presented: e.g., 0.5 mA of Ar11+ at 18 GHz, 20 μA of Ar16+ and 1.8 μA of Ar17+ when GTS is operated at 14.5 GHz. On the other hand, charge coupled device imagers have been installed to diagnose and monitor the ion be...

The HB-19 pseudopeptide 5[Kψ(CH 2 N)PR]-TASP, ψ(CH 2 N) for reduced peptide bond, is a specific inhibitor of HIV infection in different CD4 + cell lines and in primary T-lymphocytes and macrophages. It blocks virus-particle attachment to permissive cells by binding and forming a stable complex with nucleolin expressed on the cell surface. Here, we have investigated the tissue distribution of the tritiated HB-19 by using β-radio imager whole-body mapping in rats. A rapid, selective, and stable distribution and accumulation of the systematically administered HB-19 was demonstrated within the spleen, liver, bone, and kidney as soon as 5 min following its administration. No apparent uptake of HB-19 occurred in the brain and the muscle tissue. Interestingly and despite its rapid clearance from the blood, at 24 h postexposure a significant proportion of HB-19 was still recovered from target organs, of which 16–37% could be acounted for intact pseudopeptide. The elimination of HB-19 mainly occurred by renal glomerular filtration and most of the excreted radioactivity appeared to be HB-19 metabolites. Finally, injection of the biotin-labeled HB-19 pseudopeptide but not its control counterpart allowed the recovery of the HB-19–nucleolin complex from the liver, spleen, thymus, and bone marrow, thus indicating that the in vivo molecular target of HB-19 is surface nucleolin. Our results demonstrate the preferential uptake and stability of HB-19 in lymphoid organs that are the site of HIV propagation.

The availability of highly charged ion sources (electron cyclotron resonance or electron beam ion source) led in the last decade to many new scientific discoveries in various fields of atomic, solid state, and plasma physics. This article will review some of the most exciting results obtained in the field of the interaction of highly charged ions on surfaces in fundamental physics (hollow atom properties, mechanisms of electron captures and losses above, below, or at surface interactions…) as well as in applied physics (surface modifications, lithography, etc.). The deceleration and monochromatization of the ion beams delivered by the ion sources will be discussed in the framework of their use in the study of the ion surface interactions.

This talk briefly reviews the atomic properties of the hollow atoms and presents recent results on the interaction of highly charged ions with surfaces.

Functionalised carbon nanotubes are able to cross the cell membrane and to accumulate in the cytoplasm or reach the nucleus without being toxic for the cell up to 10 mM. The study clearly shows that CNTs are a very promising carrier system for future applications in drug delivery and targeting therapy.

Peptide analogues containing reversed peptide bonds between each residue along the peptide sequence (retro-inverso modification) have been analyzed for their antigenic and in vivo immunogenic properties in the MHC II and Th cell response context. Two antigenic peptides were selected for this study, namely peptide 103-115 of poliovirus VP1, which is involved in the production of Abs that neutralize the infectivity of the virus, and peptide 435-446 from the third constant region of mouse heavy chain IgG2a allopeptide gamma 2ab, which mimics a corneal Ag implicated in autoimmune keratitis. In a competition assay performed in vitro using reference hybridomas of known MHC class II restriction, both retro-inverso analogues bound (although more weakly in our test) to I-Ad and/or I-Ed class II molecules. However, in both cases, this lower affinity was apparently largely compensated in vivo, as a T cell response (with IL-2 secretion), equivalent to that obtained with the wild-type peptides, was observed following immunization of BALB/c mice with the retro-inverso analogues. Moreover, these T cells proliferated and produced IL-2 in response to the cognate peptides. It is concluded that the T cell receptors of T cells primed in vivo with the retro-inverso analogues readily cross-react with parent and retro-inverso analogue-MHC complexes. The approach of using pseudopeptides containing changes involving the backbone, and not the orientation of side chains, may thus be promising to design potent immunogens for class II-restricted T cells.

We present in this paper some experiments on the interaction of slow ${\mathrm{N}}^{6+}$, ${\mathrm{O}}^{7+}$, and ${\mathrm{Ne}}^{9+}$ ions below C or Si surfaces carried out by looking at the projectile and target x rays. The study of the x rays emitted by the ions, in contrast with studies of Auger electrons, allows the observation of a much larger part of the decay, not yet explored, below the surface. Moreover, the x rays emitted by the target atoms may identify the shell from which the electrons are captured. It is shown that the electron promotion mechanism, previously observed, which transfers, e.g., K electrons of C targets into the L shell of these ions, represents only a very small part of the interactions occurring at the first atomic layer and that the neutralization takes place, below the surface, mainly via Auger neutralization.

Slow highly charged ions penetrating surfaces quickly capture many electrons in highly excited states, leaving empty the innermost shells, forming hollow atoms. These hollow atoms then fill their innermost shells in a stepwise manner through a long cascade of Auger and x-ray transitions. We have measured the mean emission depths of the series of x rays emitted during the decay cascade of Ar hollow atoms formed below the surface of metal and dielectric materials. It has been found that the decay times of these hollow atoms are much longer in dielectrics than in metals, and at keV/$q$ kinetic energies, at depths of the order of 10--20 nm, considerably deeper than any expected value. These findings have been tentatively explained by the different responses of metals and dielectrics to the slow penetration of a highly charged ion.

We present some experimental results which demonstrate that hollow atoms (ions) can be formed above insulator surfaces, and show for the first time dramatic differences in the interactions of a given ion with a metal and a semiconductor (insulator) surface, leading to the formation of different hollow atoms (ions). These results are tentatively explained in considering the localized (valence) or delocalized (conduction) character of the captured electrons and the backscattering of the ions above insulators.

The interaction of Ar{sup (17,18)+} ions with C{sub 60} has been studied by observing coincidences between Ar {ital K} x rays and the fullerene ions and fragments. At large distances the capture of electrons from C{sub 60} into excited states of the ion has been observed and compared to the interaction of the same ions with surfaces. Most of the observed events correspond to the capture of many electrons by the ion and the loss of all but one. These results show clearly the characteristic behavior of ions flying over a surface without any contact. {copyright} {ital 1996 The American Physical Society.}

Connexin40 (Cx40) is a member of the connexin family of gap junction proteins. Its mRNA, abundant in lung, is also present in mammalian heart, although in lower amount. Rabbit antipeptide antibodies directed to the COOH terminus (residues 335 to 356) of rat Cx40 were characterized to investigate the distribution of Cx40 in rat and guinea pig cardiac tissues. The affinity-purified antibodies detect specifically a major protein (M(r), 40,000) in immunoblots of total extracts from rat lung and rat and guinea pig heart. In sections of guinea pig atrial tissue treated for immunofluorescence, a strong labeling associated with myocytes was seen with a distribution consistent with that of intercalated disks. The results of immunoelectron microscopy carried out with guinea pig atrial tissue showed that epitopes recognized by these antibodies were exclusively associated with gap junctions. These results, added to those of control experiments, demonstrate that antibodies 335-356 are specific for Cx40. Double-labeling experiments carried out with lung sections using anti-factor VIII and anti-Cx40 antibodies suggest that Cx40 is expressed in blood vessel endothelial cells. In guinea pig and rat heart sections, investigated using both immunofluorescence and immunoperoxidase techniques, a signal was also found to be associated with vascular walls. In guinea pig heart, only atrial myocytes are Cx40-positive. No labeling was detected in ventricular myocytes, including those of the His bundle and the bundle branches, which otherwise do express connexin43 (Cx43). In rat heart Cx40-expressing myocytes are localized in branches, and the Purkinje fibers. Cx43 is not detected either in the His bundle or in the proximal parts of the bundle branches, and consequently, Cx40 is the first connexin demonstrated in this region of the rat conduction system. Cx40 was not detected in the working ventricular myocytes. Double-labeling experiments carried out with hen anti-Cx43 antibodies and rabbit anti-Cx40 antibodies demonstrated that, in tissues expressing both Cx43 and Cx40, these two connexins were localized in the same immunoreactive sites. A few sites, however, appear to contain only one or the other of these two connexins.

The interaction of Ar17+, Fe25+ and Kr35+ ions on surfaces has been studied by looking at the X-rays emitted in flight by the ions with a SiLi detector. A model has been developed to describe the behavior of the ions inside the solid and the formation of “hollow atoms” below the surface.

The hyperfine-quenched transition 2 $^{3}$${\mathit{P}}_{0\char21{}}$1 $^{1}$${\mathit{S}}_{0}$ has been observed in heliumlike silver (${\mathrm{Ag}}^{45+}$) in the two isotopes $^{107}\mathrm{Ag}$ and $^{109}\mathrm{Ag}$. The lifetime for the transition has been measured for each isotope and found to be ${\mathrm{\ensuremath{\tau}}}_{0}$(107)=3.98(37)\ifmmode\times\else\texttimes\fi{}${10}^{\mathrm{\ensuremath{-}}12}$ sec and ${\mathrm{\ensuremath{\tau}}}_{0}$(109)=2.84(32)\ifmmode\times\else\texttimes\fi{}${10}^{\mathrm{\ensuremath{-}}12}$ sec. From the measured lifetimes a value is inferred for the absolute value of the 2 $^{3}$${\mathit{P}}_{0\char21{}}$2 $^{3}$${\mathit{P}}_{1}$ fine-structure splitting \ensuremath{\Vert}\ensuremath{\Delta}${\mathit{E}}_{0\mathrm{\ensuremath{-}}1}$\ensuremath{\Vert} in ${\mathrm{Ag}}^{45+}$ with the result \ensuremath{\Vert}\ensuremath{\Delta}${\mathit{E}}_{0\mathrm{\ensuremath{-}}1}$\ensuremath{\Vert}=0.79(04) eV, where the uncertainty is the experimental uncertainty taken at the 1\ensuremath{\sigma} confidence level. This result is compared with calculations based on the multiconfigurational Dirac-Fock method, the unified method, and recent results from the relativistic configuration-interaction method.

I summarize in this paper the present status of our experimental knowledge on the Lamb shift of high Z hydrogenlike ions. Some tentative prospect on the future improvements with the new large accelerators and ion sources are discussed and compared with the present accuracy of QED corrections.

The hyperfine-quenched transition 2{sup 3}{ital P}{sub 0-}1{sup 1}{ital S}{sub 0} has been observed in heliumlike gadolinium (Gd{sup 62+}) in the two isotopes {sup 155}Gd and {sup 157}Gd. The lifetime for the transition ({tau}{sub 0}) has been measured for each isotope and found to be {tau}{sub 0}(155)=13.43(27){times}10{sup {minus}12} sec and {tau}{sub 0}(157)=7.65(55){times}10{sup {minus}12} sec. From the measured lifetimes a value is inferred for the absolute value of the 2{sup 3}{ital P}{sub 0-}2{sup 3}{ital P}{sub 1} fine-structuring splitting {vert bar}{Delta}{ital E}{sub 0-1}{vert bar} in Gd{sup 62+} with the result {vert bar}{Delta}{ital E}{sub 0-1}{vert bar}=18.57(19) eV, where the error represents 1 standard deviation. This result is compared with calculations based on the multiconfiguration Dirac-Fock method and on the unified method.

The behaviour of argon hollow atoms, travelling at low velocity inside a solid target, has been studied. The filling of the K shell by the electrons of the solid screening the ions, through radiative electron capture, or co-operative electron transition, has been observed. These transitions have been found to be in competition with spontaneous decay.

Monoclonal antibodies (MAbs) were raised against native and denatured alpha-hemolysin (HlyA) of Escherichia coli. Binding of the MAbs to native, denatured, and erythrocyte-complexed active wild-type hemolysin and mutant derivatives was tested. All 15 MAbs analyzed bound to native hemolysin, even when the toxin was complexed with human erythrocytes. While some MAbs were unable to bind to a specific native mutant hemolysin, others could not even bind to mutant hemolysin carrying deletions remote from their actual binding sites. A rough determination of the binding sites of 15 MAbs on HlyA was performed by Western immunoblot analysis using CNBr fragments of HlyA and mutant hemolysin proteins. Interestingly, the binding sites of the MAbs against native hemolysin seem to be more randomly distributed on HlyA than are those of MAbs against denatured hemolysin. Three MAbs inhibited the hemolytic activity significantly. Two of these MAbs bound to the hydrophobic region, and the other one bound to the repeat domain of HlyA. The use of synthetic peptides from these regions allowed determination of the linear epitopes for two of these MAbs.

The n=2\ensuremath{\rightarrow}n=1 lines of hydrogenlike and heliumlike uranium have been observed and are being reported for the first time. The spectrum consists of all allowed and forbidden lines. We have done a preliminary energy measurement of these lines with a precision in the (4--12)\ifmmode\times\else\texttimes\fi{}${10}^{\mathrm{\ensuremath{-}}4}$ range.

Two cyclic peptide analogues corresponding to residues 139-146 (site A) of influenza A virus haemagglutinin (strain X31) were synthesized. The ability of these peptides to react with anti-influenza virus antibodies was found to depend on the conformation of the loop and on the orientation in which the peptide was presented to antibodies. Antibodies raised to the peptides were able to bind in ELISA with influenza virus antigen that had been allowed to dry on the microtitre plate. When OF1 mice were immunized with cyclic peptides, approximately 80% of the animals were protected against an intranasal challenge with influenza virus.

SUMMARY The reactivity of autoantibodies present in the serum of patients with systemic lupus erythematosus (SLE) was investigated by ELISA using seven overlapping synthetic peptides representing the entire sequence of the polypeptide D component of ‘Sm antigen’. Of the 165 SLE sera tested, 59% were found to contain IgG antibodies able to bind to peptide 1–20, while 37% of the sera reacted with peptide 44–67. All sera reacting with peptide 44–67 also reacted with peptide 1–20. These two peptides were only seldom recognized by the sera of 187 patients with other rheumatic autoimmune diseases or by 53 sera of normal individuals. In a parallel study using sera that reacted with the D band in immunoblotting, most of the sera recognized peptides 44–67 (89%) and 1–20 (67%), while 33% of them reacted with peptide 97–119. The use of these synthetic peptides in ELISA may be of considerable help for detecting anti Sm autoantibodies.

The capture of many electrons by ${\mathrm{Ar}}^{17+}$ ions, at low velocity, near a metallic surface, has been studied. Multiexcited bound states with many electrons in the outermost shells (hollow atoms) have been observed. The surrounding of an ionic excited core by many outermost electrons greatly decreases the lifetimes of the states. This characteristic decrease explains the main striking features of the relaxation of the ions.

International audience; The HIV-1 encoded apoptogenic protein Vpr induces mitochondrial membrane permeabilization (MMP) via interactions with the voltage-dependent anion channel (VDAC) and the adenine nucleotide translocator (ANT). We have designed a peptide, TEAM-VP, composed of two functional domains, one a tumor blood vessel RGD-like 'homing' motif and the other an MMP-inducing sequence derived from Vpr. When added to isolated mitochondria, TEAM-VP interacts with ANT and VDAC, reduces oxygen consumption and overcomes Bcl-2 protection to cause inner and outer MMP. TEAM-VP specifically recognizes cell-surface expressed alpha(V)beta(3) integrins, internalizes, temporarily localizes to lysosomes and progressively co-distributes with the mitochondrial compartment with no sign of lysosomal membrane permeabilization. Finally TEAM-VP reaches mitochondria of angiogenic endothelial cells to induce mitochondrial fission, dissipation of the mitochondrial transmembrane potential (DeltaPsi(m)), cytochrome c release and apoptosis hallmarks. Hence, this chimeric peptide constitutes the first example of a virus-derived mitochondriotoxic compound as a candidate to kill selectively tumor neo-endothelia.

Time‐resolved experiments (bremsstrahlung, diamagnetism, electron, and ion end‐loss currents) have been carried out at the Electron Cyclotron Resonance Ion Source Quadrumafios [A. Girard, et al., Rev. Sci. Instrum. 65, 1714 (1994)] continuously operated with heavy neutral gases. All the diagnostics reveal oscillations (on the time scale of about 1 s) which could be characterized by variations of the hot electron perpendicular temperature. The onset of this low‐frequency periodic regime with increasing heating powers and/or decreasing gas injection pressures is shown to limit the highly charged ion currents available. The addition of a lighter gas, e.g., He into Kr, delays the appearance of the periodic regime and enables one to extract higher currents.

A large high current EBIS source, working at room temperature, and using a noncryogenic solenoid, has been completed in Saclay. This source, whose current density is lower (≊100 A cm−2) than the cryogenic EBIS, has been designed to be used at very low pressures with turbo and Ti sublimator pumping. An ultimate vacuum of 8×10−12 mbar has been reached and may be obtained in a few days after opening. This ultrahigh vacuum allows very long confinement times. The electron beam has been injected and large amounts of Ar16+ and Ar17+ ions have been obtained in July. These preliminary results are in good agreement with theoretical simulations. More details about the special dynamics of the warm EBIS are presented.

Slow highly charged ions approaching surfaces at very close distances (∼nm) are known to capture many electrons in highly excited states forming hollow atoms(1)(2). These hollow atoms quickly decay to their ground states through a long cascade of autoionization processes( Auger transitions) which may be balanced , as these ions are still close to the surface, by a series of electron captures. This actual sequence of many events of capture and autoionization, alternate or not, is not experimentally known to date, and depends on the charge of the ions and of the capture processes which may be very different above metals and insulators. We review in this paper two new experiments on the kinematics of fully decelerated highly charged ions(Ar18+) above dielectric and metal surfaces, and on the behavior of ions of lower charge states (O7+ and Ne9+) above the same surfaces.

Systemic lupus erythematosus is characterised by the presence of high titers of autoantibodies reacting with various components of the small and heterogeneous nuclear ribonucleoprotein particle. It has been suggested that these antibodies are produced by an antigen-driven mechanism under the dependence of antigen-specific T cells. To investigate the role of T cell help in this process, we sought with twenty overlapping peptides the Th epitopes on the U1-70K snRNP in unprimed H-2k MRL/lpr lupus mice and immunised CBA normal mice. The peptide 131-151 was recognized by both IgG autoantibodies and CD4+ T cells from 7-9 week-old MRL/lpr mice. In this test, APCs from MRL/lpr mice were required, APCs from naive CBA mice failed to stimulate CD4+ cells from MRL/lpr mice. Peptide 131-151 bound both I-Ak and I-Ek class II molecules and favoured an IL-2 positive T cell response but not IFN-γ, IL-6 and IL-10 secretion. Segment 131-151 is localised within the RNP80 motif and contains residues that are highly conserved in many nuclear, nucleolar and cytoplasmic RNA binding proteins. In parallel, we studied the Ab response to the A2/B1 hnRNP in different murine models of lupus, and found in residues 50-70 a major epitope recognized very early during the course of the disease by Abs from most of MRL/lpr mice. Peptide 50-70 generated in CBA/J mice an effective Th cell response with IL-2 and IFN-γ secretion. Interestingly, this peptide also contains the highly conserved sequence present in peptide 131-151 of the 70K protein. It is possible that starting from a single Th epitope, the sequence of which is repeated in several self-proteins involved in the same complex or close cellular components, a larger, diversified Th response is generated, which extends via intra-and inter-molecular spreading of the T and B cell responses.

4-Amino-2-benzazepin-3-ones have proven very useful for studying the biologically active conformations of peptides. The synthesis of Pht-Aba-Xaa-OH by reaction of the corresponding 1,3-oxazolidin-5-one with trifluoromethanesulfonic acid (TFMSA) has been reported in the literature. However, when this procedure was applied to the preparation of Pht-Hba(Bn)-Gly-OH 8, many byproducts were formed and the yield of the desired aminobenzazepinones 7 and 8 was very low. We report in this paper an efficient methodology for the synthesis of Pht-Hba(2,6-Cl2-Bn)-Gly-OH 17 starting from the commercially available tyrosine. In our procedure, the dipeptide Pht-Tyr(2,6-Cl2-Bn)-Gly-OH 15 is converted to the 1,3-oxazolidin-5-one 16 which then undergoes Friedel-Crafts cyclization in the presence of tin tetrachloride to afford the desired 4-phthalimido-1,3,4,5-tetrahydro-8-(2,6-dichlorobenzyloxy)-2-be nzazepin-3-one 17 in excellent yield.

Very slow, highly charged ions impinging on metal surfaces are known to be accelerated by their image and to drop irremediably on the surfaces which they touch or slightly penetrate. We present experiments which demonstrate that above insulators or semiconductors, at normal incidence, the ions are backscattered at a certain distance from the surface and do not touch it. This finding is explained by the transient buildup of positive charges due to the removal of {ital valence} electrons, which overcome the acceleration of the ion by its own image. This effect is found to cancel out at grazing incidence. {copyright} {ital 1997} {ital The American Physical Society}

We summarize in this paper recent experiments which demonstrate, for the first time, that different kinds of hollow atoms are formed during the interaction of slow highly charged ions above and below metal, insulator and semiconductor surfaces. These results show that, in both cases, the conduction or valence character of the most weakly bound target electrons plays an essential role in the interaction. The ions are accelerated above metal surfaces (contact) and backscattered on insulators by the holes formed during the capture of valence electrons. Below the surface the feeding of the hollow atoms strongly depends on the metal or insulator character of the target and is found to be much faster in metals than in insulators.

We present a detailed analysis of the antigenic and immunogenic properties of a series of very stable peptidomimetics of a model hexapeptide corresponding to the C-terminal residues 130-135 of histone H3. Five pseudopeptide analogues of the natural sequence IRGERA were synthesized by systematically replacing, in each analogue, one peptide bond at a time by a reduced peptide bond Psi(CH2-NH). Three important features of the resulting analogues were examined. First, the analogues were tested in a biosensor system for their ability to bind monoclonal antibodies generated against the parent natural peptide, and their kinetic rate constants were measured. The results show that reduced peptide bond analogues can very efficiently mimic the parent peptide. The position of reduced bonds which were deleterious for the binding was found to depend on the antibody tested, and one monoclonal antibody recognized all five analogues. The equilibrium affinity constant toward reduced peptide bond analogues of four antibodies of IgG1 isotype induced against the parent hexapeptide was higher (up to 670 times) with certain analogues than toward the homologous peptide. Second, immunogenic properties of the five analogues were studied, and it was found that polyclonal antibodies induced against analogues in which Psi(CH2-NH) bonds were introduced between residues 130-131, 131-132, and 132-133 (R1-R2, R2-R3, and R3-R4) cross-reacted strongly with the cognate protein H3. Third, we tested the protease resistance of analogues. Altogether, the results provide a strong support for the potent applicability of reduced peptide bond pseudopeptides as components of synthetic vaccines and open a new field for the development of immunomodulatory agents.

The dynamics of the first capture and decay processes occurring during the interaction of slow highly charged ions below a surface has been studied in looking at the x rays emitted directly, or in coincidence, by impinging bare or hydrogenlike ions of various atomic numbers on solid targets. Some results on the decay processes of these hollow atoms, mainly formed below the surface, for argon, iron, and krypton ions are presented. By measuring the changes of the number of electrons in the L and M shells of the ions, compared to the lifetime of the K shell, it has been possible to evaluate the mean time for the filling of the L and M shells. These measurements are compared with a model of interaction of the ions with the surface.

The c-Jun transcription factor contains a transactivation domain that belongs to the "acidic" type of transcription activator. To determine the secondary structure of the Jun activation domain, we synthesized a peptide corresponding to amino acids 61 to 98 of c-Jun. Jun N-terminal kinases are able to phosphorylate Ser-63 and Ser-73 in vivo, which dramatically increases the transactivation potential of Jun. As this phosphorylation event may influence the secondary structure, we synthesized a second peptide containing two phosphoserine groups instead of serine in positions 63 and 73. Secondary structure predictions did not show potential for the peptides to adopt any stable, dominating conformation. Both peptides were purified and analyzed by circular dichroism spectroscopy. The peptides appeared to be flexible and essentially unstructured in aqueous solution. At acidic pH, we observed a decrease in the negative ellipticity at 202 nm, suggesting that some ordered structure might be present under these conditions. alpha-Helical conformation, as a dominating secondary structure, was induced in the presence of trifluoroethanol, and there was no significant difference between the unphosphorylated and phosphorylated peptides.

Retro-inverso peptides which contain NH-CO bonds instead of CO-NH peptide bonds are much more resistant to proteolysis than L-peptides. Moreover, they have been shown recently to be able to mimic natural L-peptides with respect to poly- and monoclonal antibodies (Guichard, G., Benkirane, N., Zeder-Lutz, G., Van Regenmortel, M. H. V., Briand, J. P., and Muller, S. (1994b) Proc. Natl. Acad. Sci. U.S.A. 91, 9765-9769). We have further tested the capacity of retro-inverso peptidomimetics to serve as possible targets for antibodies produced by lupus mice and by patients with rheumatic autoimmune diseases. Several retro-inverso peptides corresponding to sequences known to be recognized by autoantibodies were synthesized, namely peptides 28-45 and 130-135 of H3, 277-291 of the Ro/SSA 52-kDa protein, and 304-324 of the Ro/SSA 60-kDa protein, and tested with autoimmune sera by enzyme-linked immunosorbent assay. We have found that retro-inverso peptides are recognized as well as or even better than natural peptides by antibodies from autoimmune patients and lupus mice. This new approach may lead to important progress in the future development of immunodiagnostic assays, particularly in the case of diseases characterized by inflammatory reactions in the course of which the level of degradative enzymes is increased.

A series of monoclonal antibodies has been generated against an hexapeptide of sequence IRGERA corresponding to the C-terminal residues 130-135 of histone H3 and three analogues of this model peptide. The analogues correspond to the D-enatiomer, containing only D-residues, and two retro-peptides containing NH-CO bonds instead of natural amide peptide bonds. The chirality of each residue was maintained in the retro-peptide and inverted in the retro-inverso-peptide. Monoclonal antibodies were generated from mice immunized with the analogues coupled to neutral small unilamellar liposomes containing monophosphoryl lipid A as adjuvant. The reactivity of antibodies with the four analogues and with the parent protein H3 was studied in enzyme-linked immunosorbent assay and in a biosensor system. The equilibrium affinity constant (Ka) toward the retro-inverso-peptide of two out of three antibodies of IgG1 isotype induced against the L-hexapeptide was 7-75-fold higher than toward the homologous L-peptide. The range of Ka values of four antibodies of IgG1 and IgG2a isotypes generated against the retro-inverso-peptide was 0.6-1.9 x 10(9) M-1 for both the retro-inverso- and L-peptides. Furthermore, antibodies to the L- and retro-inverso-peptides cross-reacted strongly (in some cases better than with the homologous peptide) with the parent histone H3 and with chromatin subunits containing H3. The results are thus promising in respect to the potential use of retro-inverso-analogues, which are particularly stable, in the design of much more potent synthetic vaccines or to generate antibody probes.

The antigenic activity of the N-terminal region of coat protein of turnip mosaic virus (TuMV) aphid transmissible strain 1 and non-transmissible strain 31 was examined by using a panel of monoclonal antibodies (MAbs) raised against the two virus strains as well as antisera raised against several synthetic peptides from the N-terminal region of the protein. The reactivity of these antibodies was tested in ELISA and in a biosensor system (BIAcore Pharmacia) using virus particles, dissociated coat protein and synthetic peptides as antigens. Substitution of a single amino acid at position 8 in the coat protein of TuMV strain 1 abolished any cross-reactivity between MAbs to strain 1 and the substituted peptide (strain 31) in ELISA although some cross-reactivity was apparent in BIAcore inhibition experiments. In reciprocal tests with MAbs to strain 31 no cross-reactivity with the heterologous peptide was detected in either type of assay. The amino acid residue present at position 8 appears to play a critical role in the binding capacity of MAbs specific for the N-terminal region of TuMV. Antiserum to a synthetic peptide corresponding to residues 1–14 of the protein of TuMV strain 1 was found to react strongly with dissociated coat protein and intact virus particles and was able to inhibit the aphid transmission of the virus. Antiserum to the corresponding peptide of strain 31 did not have this capacity.

International audience; The Trans-activator protein (Tat) of human immunodeficiency virus (HIV) is a pleiotropic protein involved in different aspects of AIDS pathogenesis. As a number of viral proteins Tat is suspected to disturb mitochondrial function. We prepared pure synthetic full-length Tat by native chemical ligation (NCL), and Tat peptides, to evaluate their direct effects on isolated mitochondria. Submicromolar doses of synthetic Tat cause a rapid dissipation of the mitochondrial transmembrane potential (ΔΨ(m)) as well as cytochrome c release in mitochondria isolated from mouse liver, heart, and brain. Accordingly, Tat decreases substrate oxidation by mitochondria isolated from these tissues, with oxygen uptake being initially restored by adding cytochrome c. The anion-channel inhibitor 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid (DIDS) protects isolated mitochondria against Tat-induced mitochondrial membrane permeabilization (MMP), whereas ruthenium red, a ryanodine receptor blocker, does not. Pharmacologic inhibitors of the permeability transition pore, Bax/Bak inhibitors, and recombinant Bcl-2 and Bcl-XL proteins do not reduce Tat-induced MMP. We finally observed that Tat inhibits cytochrome c oxidase (COX) activity in disrupted mitochondria isolated from liver, heart, and brain of both mouse and human samples, making it the first described viral protein to be a potential COX inhibitor.

Three analogues of the model peptide of sequence IRGERA corresponding to the COOH-terminal residues 130-135 of histone H3 were synthesized, and their antigenicity, immunogenicity, and resistance to trypsin were compared to those of the natural L-peptide. The three analogues correspond to the D-enantiomer, containing only D-residues, and two retro-peptides containing NH-CO bonds instead of natural peptide bonds. The chirality of each residue was maintained in the retro-peptide and inverted in the retro-inverso-peptide. Antibodies to the four peptide analogues were produced by injecting BALB/c mice with peptides covalently coupled to small unilamellar liposomes containing monophosphoryl lipid A. Each of the four peptide analogues induced IgG antibodies of various subclasses. The IgG3 antibodies reacted similarly with the four analogues, whereas antibodies of the IgG1, IgG2a, and IgG2b isotypes showed strong conformational preferences for certain peptides. The retro-inverso-peptide IRGERA mimicked the structure and antigenic activity of the natural L-peptide but not of the D- and retro-peptides, whereas the retro-peptide IRGERA mimicked the D-peptide but not the L- and retro-inverso-peptides. The equilibrium affinity constants (Ka) of three monoclonal antibodies generated against the L- and D-peptides with respect to the four peptide analogues were measured in a biosensor system. Large differences in Ka values were observed when each monoclonal antibody was tested with respect to the four peptides. The use of retro-inverso-peptides to replace natural L-peptides is likely to find many applications in immunodiagnosis and as potential synthetic vaccines.

We report on beam-foil time-of-flight measurements of forbidden transitions in H-like and He-like Nb and Ag. The lifetimes of the 2 [sup 1][ital S][sub 0] state of He-like Nb, the 2[ital s][sub 1/2] state of H-like Ag, and the 2 [sup 3][ital P][sub 2] state of He-like Ag have been measured with precisions ranging from 1.7% to 10%. These are the highest-[ital Z] ions for which these lifetimes have been measured.

A fully automated multichannel peptide synthesizer has been constructed which performs simultaneous and rapid assembly of peptides on a 20-200 mumol scale. In situ activation of amino acids using BOP or PyBOP was chosen to give an optimized coupling chemistry. Specially designed blocks of valves, with a zero dead volume combined with an original circuitry, permit the distribution of amino acids derivatives and reagents pre-dissolved in DMF. Either Boc or Fmoc chemistry can be adapted on the synthesizer. In Boc synthesis a very rapid protocol involving Boc group deprotection in neat TFA, followed by the concomitant steps of neutralization and coupling, allows the addition of three amino acids per hour on each channel. In Fmoc chemistry we have integrated into the synthesizer an automatic TFA cleavage system that allows the peptides to be cleaved from the resin directly within the reactors used for synthesis. The stability of the Fmoc amino acid derivatives in solution in DMF was investigated, and decomposition was found to be insignificant during the time-span of a synthesis. The satisfactory performance of the instrument was demonstrated by routine synthesis of 15-20 mer peptides.

The synthesis of optically active aldehydes from N alpha-Boc-amino acids by reduction of the corresponding O,N-dimethyl-hydroxamates, while proceeding smoothly for most amino acids, has presented significant problems in the case of arginine. We demonstrate here that this difficulty can be overcome by the use of arginine derivatives in which the guanidino group is completely protected, such as in the case of N alpha-Boc-Arg(di-Z). In the case of nitro-, tosyl- or pmc-protected arginine, no satisfactory aldehyde formation can be obtained. It appears that in these cases the guanidino group is insufficiently protected and inhibits the formation of aldehydes. We also demonstrate that aldehydes can be readily obtained from N alpha-Fmoc protected amino acids.

The interaction of highly charged ions on surfaces has been studied at various incidence angles by looking at the X rays emitted in flight by the ions with a crystal spectrometer. This paper is focused on the study of the decay between the L and K shell which is mainly an Auger process for highly charged ions like Ar17+. A model has been developed to describe the behaviour of the ions outside the surface,a nd Dirac‐Fock calculations of the Auger rates for states with many electrons in high n states have been performed. The formation of highly excited hollow atoms, outside the surface, has been observed, at grazing incidence. The signature of these superexcited ions only represents a few % of the overall observed decays.In addition experiments were performed with very thin targets (20–20000 A) to study the influence of their thickness on the X ray emission. This allows a direct measurement of the lifetime of the hollow atoms inside the solid, which has been found to be less than 8 10−16 s.

Summary Eight peptides corresponding to conserved regions of the coat protein of potyviruses were synthesized. All the peptides were recognized by anti-virus or anti-core-virus. Antisera raised to the synthetic peptides were tested with purified viruses and viral antigens present in plant sap. In many cases, the extent of cross-reactivity between different potyviruses was not correlated with the degree of sequence homology between the peptide used for immunization and the corresponding region in the coat protein of the potyvirus tested. An antiserum raised to a peptide of 18 residues containing a highly conserved region was found to react with all seven potyviruses tested.