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The biologically relevant and active forms of human immunodeficiency viruses type 1 and 2 reverse transcriptase found in infectious virions are heterodimers produced in a two-step dimerization process. Dimerization involves first the rapid association of the two subunits, followed by a slow conformational change yielding a fully active form. We have shown that the dimeric nature of reverse transcriptase represents a important target for the design of a new class of antiviral agents. In this work, we propose a new strategy for its inhibition by targeting protein/protein interactions during viral formation in infected cells. From the screening of peptides derived from the tryptophan cluster at the interface of the connection subdomain, we have designed a short peptide (10 residues) corresponding to residues 395–404, which can block dimerization of reverse transcriptase in vitro and in infected cells. This peptide is highly efficient in abolishing the production of viral particles, without any adverse toxic side effects, when transduced into human immunodeficiency virus type 1-infected cells together with a new peptide carrier.

The glycolamidic ester (-O-CH2-CO-NH-) was tested as labile linkage group in solid phase peptide synthesis using a polyacrylic resin and base-cleavable fluor-enylmethoxycarbonyl (FMOC) protecting group for α NH2. Although the glycolamidic ester group was known to be highly labile in basic conditions, the 32–36 thymopoietin segment, Arg-Lys-Asp-Val-Tyr (TP5), was obtained with high yield using a FMOC-tBU strategy. The peptide was first deprotected on the carrier and then removed by saponification. The TP5 thus isolated was shown to be pure by high pressure liquid chromatography and amino acid analysis.

Gelsolin and thymosin β4 appear to be two important actin-associated proteins involved in the regulation of actin polymerization. It has been widely demonstrated that thymosin is the major cellular actin-sequestering factor shifting the polymerization equilibriuni of actin towards a monomeric state. At the same time gelsolin, a Ca2+ and inositol phosphate sensitive protein, regulates actin filament length. The interactions of these two proteins with actin are rather complex and require the participation of several complementary peptide sequences. We have identified a common motif, (I. V)EKFD, in the two proteins in the functional sequences so far examined. Gelsolin- and thymosin β4-related peptides including the common motif were synthesized and their structural and functional properties studied. These two sequences exert a major inhibitory effect on salt-induced actin polymerization. We used circular dichroism and Fourier-transform infrared spectroscopy to show that the two synthetic peptides present some secondary structure in solution. As far as the peptide derived from the thymosin sequence was concerned, α-helical structure was induced by trifluoroethanol as observed with the full-length molecule. These experiments underscore the importance of the conformational state of peptide fragments in their biological activities. ELISA and fluorescence measurements have been used to identify the binding regions of these fragments to a C-terminal region (subdomain 1) of the actin sequence. Our results also emphasize the relationship between the propensity of small sequences to form secondary structures and their propensity for biological activity as related to actin interaction and inhibition of actin polymerization. ©Munksgaard 1996.

The Rho-guanine nucleotide exchange factors (Rho-GEFs) remodel the actin cytoskeleton via their Rho-GTPase targets and affect numerous physiological processes such as transformation and cell motility. They are therefore attractive targets to design specific inhibitors that may have therapeutic applications. Trio contains two Rho-GEF domains, GEFD1 and GEFD2, which activate the Rac and RhoA pathways, respectively. Here we have used a genetic screen in yeast to select in vivo peptides coupled to thioredoxin, called aptamers, that could inhibit GEFD2 activity. One aptamer, TRIAPalpha (TRio Inhibitory APtamer), specifically blocks GEFD2-exchange activity on RhoA in vitro. The corresponding peptide sequence, TRIPalpha, inhibits TrioGEFD2-mediated activation of RhoA in intact cells and specifically reverts the neurite retraction phenotype induced by TrioGEFD2 in PC12 cells. Thus TRIPalpha is the first Rho-GEF inhibitor isolated so far, and represents an important step in the design of inhibitors for the expanding family of Rho-GEFs.

International audience; The development of peptide drugs and therapeutic proteins is limited by the poor permeability and the selectivity of the cell membrane. There is a growing effort to circumvent these problems by designing strategies to deliver full-length proteins into a large number of cells. A series of small protein domains, termed protein transduction domains (PTDs), have been shown to cross biological membranes efficiently and independently of transporters or specific receptors, and to promote the delivery of peptides and proteins into cells. TAT protein from human immunodeficiency virus (HIV-1) is able to deliver biologically active proteins in vivo and has been shown to be of considerable interest for protein therapeutics. Similarly, the third alpha-helix of Antennapedia homeodomain, and VP22 protein from herpes simplex virus promote the delivery of covalently linked peptides or proteins into cells. However, these PTD vectors display a certain number of limitations in that they all require crosslinking to the target peptide or protein. Moreover, protein transduction using PTD-TAT fusion protein systems may require denaturation of the protein before delivery to increase the accessibility of the TAT-PTD domain. This requirement introduces an additional delay between the time of delivery and intracellular activation of the protein. In this report, we propose a new strategy for protein delivery based on a short amphipathic peptide carrier, Pep-1. This peptide carrier is able to efficiently deliver a variety of peptides and proteins into several cell lines in a fully biologically active form, without the need for prior chemical covalent coupling or denaturation steps. In addition, this peptide carrier presents several advantages for protein therapy, including stability in physiological buffer, lack of toxicity, and lack of sensitivity to serum. Pep-1 technology should be extremely useful for targeting specific protein-protein interactions in living cells and for screening novel therapeutic proteins.

The primary amphipathic peptide Ac-Met-Gly-Leu-Gly-Leu-Trp-Leu-Leu-Val-Leu10-Ala-Ala-Ala-Leu-Gln-Gly-Ala-Lys-Lys-Lys20-Arg-Lys-Val-NH-CH2-CH2-SH called SPM was able to induce formation of ion channels into planar lipid bilayers with main conductance values of 75 and 950 pS in 1 KCl. The 75 pS value can be attributed to an aggregate composed of five monomers since the corresponding five-unit bundle (5-SPM) also presented a 70 pS channels under the same conditions. The upper 950 pS level would be generated by a hexameric aggregate. Ion channels induced by both SPM and its pentameric bundle are slightly cation selective but not voltage-dependent. The structural studies showed that the SPM and 5-SPM possess mainly an α-helical structure (∼40%) and are strongly embedded in the bilayer. This behaviour and the strong hydrophobic interactions occurring between helices in the bundle induce a strong stabilization of 5-SPM in the bilayer and would be responsible for the stepwise current fluctuations observed during the incorporation of 5-SPM into the membrane. Copyright © 2001 European Peptide Society and John Wiley & Sons, Ltd.

International audience; We have shown previously that a peptide, MPG, derived from the hydrophobic fusion peptide of HIV-1 gp41 and the hydrophilic nuclear localisation sequence of SV40 large T antigen, can be used as a powerful tool for the delivery of oligonucleotides into cultured cells. Now we extend the potential of MPG to the delivery of nucleic acids into cultured cells. In vitro, MPG interacts strongly with nucleic acids, most likely forming a peptide cage around them, which stabilises and protects them from degradation in cell culture media. MPG is non-cytotoxic, insensitive to serum and efficiently delivers plasmids into several different cell lines in only 1 h. Moreover, MPG enables complete expression of the gene products encoded by the plasmids it delivers into cultured cells. Finally, we have investigated the potential of MPG as an efficient delivery agent for gene therapy, by attempting to deliver antisense nucleic acids targeting an essential cell cycle gene. MPG efficiently delivered a plasmid expressing the full-length antisense cDNA of human cdc25C, which consequently successfully reduced cdc25C expression levels and promoted a block to cell cycle progression. Based on our results, we conclude that MPG is a potent delivery agent for the generalised delivery of nucleic acids as well as of oligonucleotides into cultured cells and believe that its contribution to the development of new gene therapy strategies could be of prime interest.

We describe the design and the Fmoc/tBu solid phase synthesis of a 20 residue long peptide containing five regularly distributed lysines. Cyclization of this peptide was achieved using BOP as coupling agent. After side-chain deprotection, all the basic residues were iodoacetylated and then allowed to react either with a C-terminal free COOH peptide or with peptides bearing a cysteamide group. The final pentameric templates were identified by mass and amino acid analysis which gave data compatible with the expected values. Copyright © 1999 European Peptide Society and John Wiley & Sons, Ltd.

The conformations of two peptides produced by the combinations of a nuclear localization sequence and a sequence issued from the fusion protein gp41 of HIV 1 have been analyzed both in solution and in membranes or in membrane mimicking environments. Both are shown to be nonordered in water, alpha-helical when incorporated into SDS micelles where the helical domain concerns the hydrophobic part of the peptides. Interactions with lipids induce the formation of beta-sheet and the lipid-peptide interactions are governed by the nature of the lipid polar headgroups. A monolayer study shows that replacement of the sequence separating the two sequences with an arginine favors the lipid-peptide interactions which may contribute to the understanding of the different, nuclear and membrane associated, cellular localizations of the peptides.

The cytotoxic properties of an amphiphilic synthetic peptide are presented. Comparative analysis of proliferating, differentiated and confluent H9C2 adherent cells and L1210 cells in suspension shows a correlation between toxicity and cell stage (proliferating cells). Electrophysiological measurements on Xenopus laevis oocytes bathed in the peptide also demonstrated the induction of cationic currents, which is voltage and phosphate dependent. These results allow us to hypothesize that the observed toxicity is related to membrane hyperpolarization of proliferating cells at the G1/S cell cycle phase transition.

Cyclic acylurea readily formed from Dpr(Phoc) linker was shown to provide linkage that is fully compatible with standard Fmoc-based solid-phase peptide synthesis protocols. The utility of this linkage was checked during Tyr-Asp-Pro-Ala-(Pro) 6 -OH synthesis which is very prone to diketopiperazine formation at the dipeptide stage. This side-reaction was avoided by maintaining the Dpr(Phoc) linker until the tripeptide stage, while piperidine had to be temporary replaced with morpholine as reagent for Fmoc group removal.

In vertebrates, unfertilized eggs are arrested at second meiotic metaphase by a cytostatic factor (CSF), an essential component of which is the product of the c-mos proto-oncogene. CSF prevents ubiquitin-dependent degradation of mitotic cyclins and thus inactivation or the M phase-promoting factor (MPF). Fertilization or parthenogenetic activation triggers a transient increase in the cytoplasmic free Ca2+ (reviewed in refs 5 and 6), inactivates both CSF and MPF, and releases eggs from meiotic metaphase arrest. A calmodulin-dependent process is required for cyclin degradation to occur in cell-free extracts prepared from metaphase II-arrested eggs (CSF extracts) when the free Ca2+ concentration is transiently raised in the physiological micromolar range. Here we show that when a constitutively active mutant of calmodulin-dependent protein kinase II (CaM KII) is added to a CSF extract, cyclin degradation and Cdc2 kinase inactivation occur even in the absence of Ca2+, and the extract loses its ability to cause metaphase arrest when transferred into embryos. Furthermore, specific inhibitors of CaM KII prevent cyclin degradation after calcium addition. Finally, the direct microinjection of constitutively active CaM KII into unfertilized eggs inactivates Cdc2 kinase and CSF, even in the absence of a Ca2+ transient. The target for Ca(2+)-calmodulin is thus CaM KII.

International audience; Studies on the externalities of landfills emphasize the specific difficulties to evaluate the environmental cost of leachate leakage, due to several uncertainties (durability of liners on a secular timescale, value society puts on groundwater quality and on its own future through the discounting procedure used in cost-benefit analysis ). Then, while published external costs range from 0¤/t to 2¤/t, opponents to landfill projects often argue on the potential pollution of the underlying groundwater for refusing such installations, which could mean that such low cost figures do not represent at all the real social value of this environmental risk. We first present the composition of leachate from eight French municipal solid waste landfills, focusing on organic micro-pollutants some of which had not been previously encountered or looked at in France. From those data we have identified that some compounds such as toluene and bisphenol A should be especially studied in risk evaluation. The potential concentrations in the groundwater are then quantified using a numerical model of transfer through current landfill liners including diffusion processes of organic contaminants in synthetic and mineral materials. It is generally below the health limits for the two compounds for which calculations where performed: benzene and dichloromethane. Last, we propose a socio-economic interpretation on the environmental risks linked to the quality of barriers, including: - Intergenerational equity considerations through an original discounting procedure (generation-adjusted discounting), - spatial equity through the territorial consequences of the potential application of the current French guide on the design on landfill liners based on the concept of equivalent level of protection. The cost of using high quality liners in landfills may be then better justified, leading to more sustainable ways of designing and locating final sinks.

In order to obtain a peptide retaining its biological activity following microinjection into living cells, we have modified a synthetic peptide [PKi(m)(6-24)], derived from the specific inhibitor protein of the cAMP-dependent protein kinase (A-kinase) in two ways: (1) substitution of the arginine at position 18 for a D-arginine; (2) blockade of the side chain on the C-terminal aspartic acid by a cyclohexyl ester group. In an in vitro assay, PKi(m) has retained a specific inhibitory activity against A-kinase as assessed against six other kinases, with similar efficiency to that of the unmodified PKi(5-24) peptide. Microinjection of PKi(m) into living fibroblasts reveals its capacity to prevent the changes in cell morphology and cytoskeleton induced by drugs which activate endogenous A-kinase, whereas the original PKi peptide failed to do so. This inhibition of A-kinase in vivo by PKi(m) lasts between 4 and 6 h after injection. In light of its effective half-life, this modified peptide opens a route for the use of biologically active peptides in vivo, an approach which has been hampered until now by the exceedingly short half-life of peptides inside living cells. By providing a direct means of inhibiting A-kinase activity for sufficiently long periods to observe effects on cellular functions in living cells, PKi(m) represents a powerful tool in studying the potential role of cAMP-dependent phosphorylation in vivo.

The technical rules of municipal solid waste landfills require liners against leachate, allowing a partial internalisation of the environmental externalities. Nevertheless, fugitive leachate flows remain due to hydraulical, mechanical and chemical stresses on liners. To quantify the remaining externalities, a monetarized risk evaluation was carried out from international bibliographical data and original French field data on leachate composition, especially the micro-pollutants, by modelling the advective and diffusive processes flow through liners, then by an economic analysis of the impacts of the computed concentrations. The original data on leachate from French landfills confirm for the first time in France the presence of specific micro-pollutants. The modelling of the transfer of two specific pollutants shows the relevance of some specific engineering of liners. The economic analysis of damages shows that, owing to the assumptions made, the current threshold for benzene and methyl chloride concentrations for health and the environment can be respected at a reasonable cost. Finally, the purely technical concept of liner equivalence is suggested to be enriched by socioeconomic considerations.; La réglementation des installations de stockage de déchets ménagers et assimilés impose des barrières d'étanchéité aux lixiviats, permettant ainsi une internalisation partielle des externalités environnementales. Il reste toutefois un débit de fuite dû aux sollicitations hydrauliques, mécaniques et chimiques des barrières. Pour quantifier ces externalités résiduelles, une évaluation monétarisée des risques a été entreprise à partir de données bibliographiques internationales et de données de terrain françaises originales sur la composition des lixiviats, en particulier en micro-polluants, en modélisant les transferts à travers les barrières par advection et diffusion, puis en faisant une analyse économique des impacts des concentrations ainsi calculées. Les données originales sur la composition chimique des lixiviats confirment pour la première fois en France la présence de certains micro-polluants. La modélisation des transferts de deux de ces polluants a mis en évidence l'intérêt de certaines dispositions constructives des barrières. L'analyse économique des impacts montre qu'avec les hypothèses de calcul prises, les seuils actuels d'acceptabilité sanitaire et environnementale pour le benzène et le dichlorométhane peuvent être respectés à un coût raisonnable. On suggère enfin d'enrichir la notion purement technique d'équivalence des barrières par des considérations socioéconomiques.

Glycolamidic ester linkage of a peptide chain to a polystyrene resin can be easily cleaved under smooth alkaline conditions.

Two families of primary amphipathic peptides issued from the association of a nuclear localization sequence (NLS) with either a signal (SP) or fusion peptide (FP) were shown to be efficient carriers of drugs or nucleic acids [1,2]. In order to define the minimal structural requirements that maintain the vector properties, the identification of the membrane crossing process was a crucial step. Here, we focus on the uptake of the vectors by model membranes and examine the influence of the hydrophobic sequence, and thus of its conformation, on the penetration into phospholipid monolayers of various physical states and polar headgroups.

International audience; Human cdc25C is a dual-specificity phosphatase involved in the regulation of cell cycle progression in both unperturbed cells and in cells subject to DNA damage or replication checkpoints. In this study, we describe the structure-function relationship of an essential domain of human cdc25C that interacts with 14-3-3 proteins. We show that this domain is a bi-functional interactive motif that interacts with cyclins primarily through their P-box motif in addition to 14-3-3 proteins. Characterization of the structural features of this domain by NMR and circular dichroism reveals two distinct alpha helical moieties interconnected by a loop carrying the 14-3-3 binding site. Moreover, the helical folding is induced upon binding to 14-3-3, suggestive of a conformational regulation of this domain of cdc25C through interactions with partner proteins in vivo. Combining our structural and biochemical data, we propose a detailed model of the molecular mechanism of cdc25C regulation by differential association with 14-3-3 and cdc2-cyclin B.

International audience; The biologically relevant and active form of human immunodeficiency virus reverse transcriptase is a heterodimer produced in a two-step dimerization process. Dimerization involves first the rapid association of the two subunits, followed by a slow conformational change yielding a fully active form. In the present study, we demonstrate that the interaction between the thumb domain of p51 and the RNase-H domain of p66 plays a major role in an essential conformational change required for proper folding of the primer/template and the tRNA-binding site, for maturation and for activation of heterodimeric reverse transcriptase. A synthetic peptide derived from the sequence within the thumb domain of p51, which forms the interface with the RNase-H domains of p66, binds heterodimeric reverse transcriptase with an apparent dissociation constant in the nanomolar range and selectively inhibits activation of heterodimeric reverse transcriptase with an inhibition constant of 1.2 microM. A detailed study of the mechanism of inhibition reveals that this peptide does not require dissociation of heterodimeric RT for efficient inhibition and does not affect subunit association, but interferes with the conformational change required for activation of heterodimeric reverse transcriptase, resulting in a decrease in the affinity of reverse transcriptase for the tRNA and an increase in the stability of the primer/template/reverse transcriptase complex. We have previously proposed that the dimeric nature of reverse transcriptase represents an interesting target for the design of antiviral agents. On the basis of this work, we propose that the conformational changes involved in the activation of reverse transcriptase similarly represent an important target for the design of novel antiviral compounds.

International audience; We have designed, synthesized and purified a 51 amino acid peptide derived from an essential domain of human cdc25C phosphatase. In vivo, differential phosphorylation of this domain regulates either the induction of mitotic processes, or the checkpoint arrest of eukaryotic cells in response to DNA damage. Peptide synthesis was achieved using the stepwise Fmoc strategy and resulted in an important yield of highly pure peptide. The final peptide was identified by amino acid analysis, electrospray mass spectrometry and nuclear magnetic resonance, which revealed that one of the two methionines within the peptide was oxidized into its sulphoxide derivative We investigated whether this 51 amino acid peptide folded into secondary structures in solution by circular dichroism and observed the formation of alpha helices in TFE. Finally, we verified that this peptide could bind to its biologically relevant 14-3-3 partner in vitro by fluorescence spectroscopy.

A peptide nucleic acid (PNA) antisense for the AUG translation initiation region of prepro-oxytocin mRNA was synthesized and coupled to a r etro-inverso peptide that is rapidly taken up by cells. This bioconjugate was internalized by cultured cerebral cortex neurons within minutes, according to the specific property of the vector peptide. The PNA alone also entered the cells, but more slowly. Cell viability was unaffected when the PNA concentrations were lower than 10 microM and incubation times less than for 24 h. Magnocellular neurons from the hypothalamic supraoptic nucleus, which produce oxytocin and vasopressin, were cultured in chemically defined medium. Both PNA and vector peptide-PNA depressed the amounts of the mRNA coding for prepro-oxytocin in these neurons. A scrambled PNA had no effect and the very cognate prepro-vasopressin mRNA was not affected. The antisense PNA also depressed the immunocytochemical signal for prepro-oxytocin in this culture in a dose- and time-dependent manner. These results show that PNAs driven by the retro-inverso vector peptide are powerful antisense reagents for use on cells in culture.

The structure and conformational behaviour of a vector peptide, designed by association of a fusion peptide and a nuclear localization sequence, are described. A beta-sheet domain is observed in which fluorescence measurements show that ten peptide molecules bind one all-trans retinol or all-trans retinoic acid molecule with a strong affinity (K'd = 40 nM). Stoichiometry and affinity of the binding can be compared with those of cellular retinoid binding proteins, the structure of which is an anti-parallel beta barrel. Analogy between the system under study and cellular retinoid-binding proteins is discussed. Peptide-helped internalization and subsequent perinuclear localization of retinol in human fibroblast cells confirm this analogy. Also, this last result shows that the peptide is an efficient carrier for insoluble substances like retinoids.

Peptides containing a hydrophobic motif associated with a nuclear localization signal separated by various linkers were synthesized in solid phase. The hydrophobic sequence corresponds either to a signal peptide sequence or to a fragment of the fusion peptide of GP41 while the hydrophilic sequence is that of a nuclear localization signal. The C-termini of these peptides bear a cysteamide group that was linked to a fluorescent probe. This allowed the cellular localization of the probe to be determined as a function of the peptide sequences. The labeled peptides were then incubated with fibroblasts. Using N-biotinylated derivatives we confirmed by indirect immunofluorescence that the observed localizations corresponds to those of the peptides. The presence of a linker appears to play a role in the cellular localization. One of these peptides was successfully used to target fluorescent oligodeoxynucleotides into living cells demonstrating improved cell delivery of peptide-oligodeoxynucleotide conjugates.

Two peptides designed for drug delivery were generated by the combination of a signal peptide with a nuclear localization sequence and are shown to facilitate the cellular internalization of small molecules which are covalently linked to these peptides. In order to understand the mechanism of internalization, the conformations of the peptides were investigated through different approaches both in solution and in membrane-mimicking environments. These peptides are highly versatile and adopt different conformational states depending on their environment. While in a disordered form in water, they adopt an alpha-helical structure in TFE and in the presence of micelles of SDS or DPC. The structured domain encompasses the hydrophobic part of the peptides, whereas the charged C-termini remain unstructured. In contrast, in the presence of lipids and whatever the nature of the phosphate headgroup, the two peptides mainly adopt an antiparallel beta-sheet form and embed in the lipidic cores. This result suggests that the beta-sheet is responsible for the translocation through the cellular membranes but also questions the conformational state of signal peptides when associated to hydrophilic sequences.

Gelsolin, a calcium and inositol phospholipid-sensitive protein, regulates actin filament length. Its activity is complex (capping, severing, etc.) and is supported by several functional domains. The N-terminal domain alone (S1), in particular, is able to impede actin polymerization. Our investigations were attempted to precise this inhibitory process by using synthetic peptides as models mimicking gelsolin S1 activity. Three peptides issued from S1 and located in gelsolin-actin interfaces were synthesized. The peptides (15-28, 42-55, and 96-114 sequences) were tested for their conformational and actin binding properties. Although the three peptides interact well with actin, only peptide 42-55 affects actin polymerization. A detailed kinetic study shows that the latter peptide essentially inhibits the nucleation step during actin polymerization. In conclusion, the present work shows that the binding of a synthetic peptide to a small sequence located outside the actin-actin interface is essential in the actin polymerization process.

The use of disulfide bonds for peptide-resin linkage in solid-phase peptide synthesis was investigated using polyacrylic polymers (Expansin) and automated Fmoc methodology. The disulfide moiety was bound to the support either by coupling a protected bifunctional handle or by an original stepwise procedure. Among the three different disulfide handles that were investigated, only the aminoethyldithio-2-isobutyric acid (AEDI) handle was stable enough to achieve peptide synthesis. A series of peptides of up to 10-20 amino acids were prepared in this manner, in good yield and purity. Rapid and quantitative peptide release was obtained by reduction with equimolecular amounts of dithiothreitol at pH 9 or tris(2-carboxymethyl) phosphine at pH 4.5. This allowed direct and rapid coupling of the released cysteamide peptides to an activated protein carrier and the use of free or resin-bound forms of the antigen in immunoassays.

With the aim of developing active protection against the noxious effects provoked in humans by scorpion stings, the possibility of eliciting toxin reactive antibodies by immunization with a short peptide was assessed in mice. The amino acid sequence of residues 50 to 59 of Androctonus australis Hector toxin II was chosen, on the basis of previous results indicating that rabbit anti-(50-59) antibodies neutralize the biological effects of the parent toxin. The peptide was prepared by solid-phase synthesis procedures and used in different forms (free, linearly polymerized, coupled to KLH, coupled to a low-molecular weight B-lymphocyte activator) in order to immunize groups of non-congenic NMRI or congenic C57BL/6 mice. The reactivities of each serum with the peptide and with the toxin were assessed in ELISA. Strong reactivities with both the peptide (mean titer over 1:52,600) and the toxin (mean titer 1:800) were observed in all mice from the group that received the KLH-coupled peptide. However, mouse immune sera failed either to recognize the toxin in a liquid-phase radioimmunoassay or to neutralize the lethal effects of the toxin. The requirements, in terms of affinity and recognition of native conformation, for anti-peptide antibodies to display neutralizing properties are discussed.

The solid-phase synthesis of the octapeptide 1 AcGly-Ala-Lys-Arg-His-Arg-Lys-ValOMe, which represents the fragment 14-21 of the amino acid sequence of the chromosomal histone H4, as well as of the structurally related nonapeptide 2 AcGly-Ala-Lys-Leu-Arg-His-Arg-Lys-ValOMe, is described using a new polyacrylic resin containing a glycolamide ester linkage(resin-NHCO-CH 2 -OCO-peptide) acting as a labile anchoring moiety between the resin and the peptide. After elongation of the polypeptide chain using classical protecting groups, i.e. t-butyloxycarbonyl for the α-NH 2 function, benzyloxycarbonyl, nitro and 2,4-dinitrophenyl groups for the side-chains of Lys, Arg and His respectively, both peptides 1 and 2 were obtained in good yields and with a high purity as shown by high-pressure liquid chromatography, by amino-acid analysis and by high-field proton NMR spectroscopy. This work demonstrates the ability of the newly introduced polyacrylic resin to act as a convenient support for solid-phase peptide synthesis.

The enzymatic acetylation of e-NH2 groups of lysyl residues of histones may be important in the control of the structure and the function of chromatin [ 11. All the histones that make up the chromatin core particle, i.e., histones H2A, H2B, H3 and H4, are subject to enzymatic acetylation [2]. Primary structure studies of histones show that only a few specific lysyl residues are involved and that the extent of acetylation depends on cellular activity [2-41. The chemical modification of a specific lysyl residue by acetylation has been described as a molecular process which is able to regulate the structure of DNA in chromatin [5]. On the basis of sequence analogies, a classification into two types has been proposed [2] for the acetylation sites of histones: (i) Type A with a single lysyl residue surrounded by two non-basic residues; (ii) Type B comprising a basic doublet such as LysLys,Lys-Arg and Arg-Lys. This classification suggests that only a small fragment of the histone molecule is necessary for enzymatic recognition. This work shows that an acetyltransferase acting on histone H4 is also able to acetylate the synthetic octapeptide AcCly-Ala-Lys-Arg-His-Arg-LysValNH? which corresponds to the sequence fragment of histone H4 between residues 14 and 2 1 and includes two lysyl residues, one of which Lys16 is the major site of acetylation in calf thymus histone H4. Peptide [ 14-2 l] contains two basic doublets, i.e., Lys16Arg and Arg-Lys”, which correspond to acetylation sites of type B. The ability of the acetyltransferase to recognize either both sites or one of them selectively has been investigated.

Three basic hexapeptides which have two adjacent arginyl residues were studied by field desorption mass spectrometry. At low emitter temperature singly and doubly charged pseudomolecular ions [M + 1]+ and [M + 2]2+ were observed. At higher temperature, these peptides gave a relatively complex fragmentation pattern which can be explained as resulting from preferential cleavages involving the Arg-Arg sequence. A mechanistic interpretation is proposed for this type of fragmentation.

Acetylation and deacetylation of the ~-amino groups of specific histone lysyl residues occurs in the course of the cell cycle. The overall level of acetylation at any given time involves an equilibrium between acetate uptake catalysed by nuclear acetyltransferases [ 1,2] and acetate removal catalysed by nuclear deacetylases [3-51. All the histones that make up the chromatin core particle, i.e., histones H2A, H2B, H3 and H4 are subject, in vivo, to acetylation and deacetylation but no acetylation sites have been observed on the very lysine-rich histone Hl [2]. On the basis of sequence analogies, a classification of the acetylation sites into two types (A,B) was proposed [2]. In type A there is a single lysyl residue surrounded by two non-basic residues and in type B a basic doublet such as Lys-Lys, Lys-Arg and ArgLys. This classification suggests that only a small fragment of the histone molecule is necessary for enzymatic recognition. However, chemical studies on the activity of a calf thymus deacetylase using histone H4 fragments, including the monoacetylated peptide (15-2 1) H-Ala-Lys”j (Ac)-Arg-His-Arg-LysVal-OH, led to the conclusion that a rather long sequence of the histone molecule is required for enzyme recognition [6]. Here we report the enzymatic deacetylation of the diacetylated histone H4 fragment (14-21) Ac-GlyAla-Lys-(AC)-Arg-His-Arg-Lys(Ac)-Val-NH?, with the Nand C-terminal groups protected by an acetyl and an amide group, respectively, in conditions similar to those used for the deacetylation of the whole histone H4 molecule. 2. Materials and methods

(D-Trp)6-LHRH:pGlu-His-Trp-Ser-Tyr-D-Trp-Leu-Arg-Pro-GlyNH2 was prepared by solid-phase peptide synthesis using the nitro group to protect the guanidine side chain of the arginyl residue. Removal of the side-chain protecting groups was carried out by catalytic transfer hydrogenation (CTH) using palladium acetate/ammonium formate or palladium on charcoal/formic acid. We show in this paper that this deprotection method induces i) reduction of the tryptophan residue and ii) epimerization at the histidine level (with palladium acetate/ammonium formate). Despite the formation of significant amounts of reduced peptide, CTH enabled us to obtain (D-Trp)6-LHRH in relatively good yield.

Rat liver chromatin core particles digested with clostripain yield a structurally well-defined nucleoprotein particle with an octameric core made up of fragmented histone species (designated H'2A, H'2B, H'3 and H'4, respectively) after selective loss of a sequence segment located in the N-terminal region of each core histone. Sequential Edman degradation and carboxypeptidase digestion unambiguously establish that histones H2A, H2B, H3 and H4 are selectively cleaved at the carboxyl side of Arg 11, Lys 20, Arg 26 and Arg 19 respectively and that the C-terminal sequences remain unaffected. Despite the loss of the highly basic N-terminal regions, including approximately 17% of the total amino acids, the characteristic structural organization of the nucleosome core particle appears to be fully retained in the proteolyzed core particle, as judged by physicochemical and biochemical evidence. Binding of spermidine to native and proteolyzed core particles shows that DNA accessibility differs markedly in both structures. As expected the proteolyzed particle, which has lost all the in vivo acetylation sites, is not enzymatically acetylated, in contrast to the native particle. However, proteolyzed histones act as substrates of the acetyltransferase in the absence of DNA, as a consequence of the occurrence of potential acetylation sites in the core histones thus rendered accessible. The possible role of the histone N-terminal regions on chromatin structure and function is discussed in the light of the present observations with the new core particle obtained by clostripain proteolysis.

Summary The paper reports a thermal denaturation analysis of calf thymus DNA associated with synthetic basic peptides (di-, tri-, and tetra-) including the X-X structure (basic doublet, X = Arg of Lys) which appears to be characteristic of the primary structure of all the known histones. Analogies in thermal behaviour between peptides with a basic doublet structure and aliphatic diamines of the series H 2 N (CH 2 ) n NH 2 , suggest that a positive doubly charged structure of defined length is crucial for the interaction with double stranded DNA. A model is proposed for the interaction between histones and DNA on the basis of the above-mentioned intercharge distance requirement (diamine model), which is detailed in the case of the arginine-rich histone F2a1. The role of the enzymatic acetylation and deacetylation of specific lysine residues in the case of arginine-rich histones is discussed in terms of the described diamine model. A possible role of the residue His 18 of histone F2a1 is considered in connection with this model. Proton magnetic resonance studies of DNA associated with two model compounds, malouetine (a steroidal bis quaternary ammonium salt) and histidinamide, demonstrate that accurate description (magnetic, thermodynamic and kinetic) of the complexes can be obtained. Preliminary results, using the analytical ultracentrifugation technique, in the case of some diamine complexes with circulary closed PM2 DNA are also reported.

We describe the solid phase synthesis of an amphipathic peptide C-terminated by a cysteamide group which allows further addition after removal from the resin and cleavage of the side-chain protecting groups. The peptide is shown to be rapidly internalized by cells with a nuclear localization of the peptide. When the peptide is linked to an oligonucleotide, the conjugate is also internalized with a final localization that is mainly cytoplasmic.

International audience; The more and more widespread use of geosynthetic barriers has reduced the leachate leakage from landfills, but many authors stress that there are remaining flows due to defects and diffusion processes through aged geosynthetics. A French research program (DURAGEOS) aiming at knowing the detailed physics, chemistry and (possibly) biology of the ageing processes of geomembranes and geosynthetic clay liners took place recently. One of the objectives was to know the consequences of geosynthetics ageing on leachate leakage from landfills. A focus was made on the transfer of phenolic compounds since these substances are harmful and their transfer properties unknown in geosynthetics. Diffusion coefficient through HDPE geomembranes and geosynthetic clay liners were especially measured for the first time. The environmental impacts could then be evaluated by the use of a numerical 1D model combining advection, diffusion and dispersion through different generic barriers and the groundwater. Results will be presented in this paper. Calculated concentrations will be discussed from the viewpoint of ecosystems and human health, taking into account the existing dose response relationships and the specific socioeconomic valuation problems linked to the intergenerational temporal horizons involved. Last but not least, some technical recommendations about the choice and the use of geosynthetics for safer final sinks are suggested.

We describe the synthesis of an amphipathic vector peptide which is able to form complexes with nucleic acids. Based on circular dichroism investigations, the nature of the structure obtained in water is questioned. The peptide adopts an α-helical structure in TFE and is partially in a β-sheet conformation in phosphate buffer at low peptide concentrations.

We report the solid-phase synthesis by the Fmoc strategy of a peptide containing a cysteamide group at its C-terminus. This peptide was subjected to further modifications including the linkage of fluorophores, namely lucifer yellow and coumarin respectively, at the C- and/or N-terminals. After incubation with living cultured cells these two probes were localized and it is concluded that the post-synthesis modifications can strongly modify the localization of the peptide.