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T Montenay-Garestier

Publications and source records attributed to T Montenay-Garestier.

At least 37 records · Page 2Linked to original sources
PubMed1987

Role of tyrosine and tryptophan residues in the structure-activity relationships of a cardiotoxin from Naja nigricollis venom.

This paper is an attempt to localize the critical area determining toxicity in a snake cardiotoxin. Toxin gamma is a single-chain polypeptide of 60 amino acids, which has been isolated from the venom of the African spitting cobra, Naja nigricollis. Three aromatic residues, namely, Trp-11, Tyr-22, and Tyr-51, have been individually modified by chemical means. The structure of the native toxin and of each derivative has been carefully investigated by circular dichroism, fluorescence, proton magnetic resonance spectroscopy, and two specific monoclonal antibodies. None of the chemical modifications alters the overall structure of the toxin, which in all cases remains folded into three adjacent loops (I, II, and III) rich in beta-pleated sheet emerging from a small globular region containing four disulfide bridges. A number of subtle changes, however, have been detected in the structure of each derivative compared with that of the native toxin. In particular, nitration of Tyr-51 provoked a structural perturbation in the globular region. Nitration of Tyr-22 induces a more substantial change in the beta-sheet area of the molecule. Thus, the strong inter-ring NOE that is observed in the native toxin between Tyr-22 and Tyr-51 vanishes in the Tyr-22 derivative, and significant changes are observed in the globular region. In contrast, no alteration of the beta-sheet structure of loops II and III has been detected after modification of Trp-11. All changes observed for this derivative remain located in the vicinity of the indole side chain of Trp-11 in loop I. The biological consequences of the modifications were measured: the lethal potency in vivo in mice and the cytotoxic activities in vitro on FL-cells. Lethal activities correlate with cytotoxicity: Tyr-51 modified toxin is equally potent as native toxin, whereas Tyr-22 and Trp-11 derivatized toxins are characterized by substantially lesser activities, the Trp-11 derivatized toxin being the least potent. We conclude that (1) Tyr-51 is not involved in the functional site of the toxin, although it is in interaction with the core of the molecule, (2) Tyr-22 may play a dual structural and functional role, and (3) Trp-11 is in, or in close proximity to, the functional site of the toxin. These data indicate the importance of loop I in determining toxicity of the cardiotoxin.

Animals↗
PubMed1987

Does tryptophan intercalate in DNA? A comparative study of peptide binding to alternating and nonalternating A.T sequences.

The interactions of a tetrapeptide, lysyltryptophylglycyllysine tert-butyl ester (KWGK), with synthetic double-stranded polynucleotides [poly(dA).poly(dT), poly[d(A-T)], poly(rA).poly(dT), and poly(rA).poly(rU)], Escherichia coli DNA, and single-stranded polynucleotides [poly(rA), poly(rU), poly(dA), and poly(dT)] were studied in a low-salt buffer by absorption and fluorescence spectroscopy. From fluorescence quenching data, we determined the two binding constants K1 and K2 of the two-step mechanism previously proposed for lysyltryptophyllysine binding to polynucleotides [Brun, F., Toulmé, J.J., & Hélène, C. (1975) Biochemistry 14, 558-563]. The first complex (PN)1 is purely due to electrostatic interactions between the lysyl residues and the phosphate groups. The second complex (PN)2 involves an additional stacking of the indole moiety of the tryptophyl residue with the bases (or base pairs) of the polynucleotide and is in equilibrium with (PN)1. K2 measures the ratio of the concentrations of stacked and unstacked complexes. The fluorescence decay of the tryptophyl residue in KWGK was not significantly different in the presence and in the absence of double-stranded polynucleotides in agreement with the previous model, which assumes total quenching of tryptophan fluorescence in complex (PN)2 and identical fluorescence characteristics for free KWGK and complex (PN)1. The stacking of the tryptophyl residue with A.T base pairs in alternating poly[d(A-T)] was found to be 10 times more efficient than that with nonalternating poly(dA).poly(dT). Among A-T-containing double-stranded polynucleotides, poly(rA).poly(dT) was found to be the most favorable for tryptophan stacking. A similar behavior was previously demonstrated for several intercalating agents such as ethidium bromide, propidium iodide, and daunomycin.(ABSTRACT TRUNCATED AT 250 WORDS)

DNA↗
PubMed1987

Oligo-[alpha]-deoxynucleotides covalently linked to an intercalating agent. Double helices with parallel strands are formed with complementary oligo-[beta]-deoxynucleotides.

An oligo-[alpha]-deoxynucleotide of sequence (5')d(TCTAAACTC) (3') was synthesized using the alpha-anomers of deoxynucleosides and its 5'-phosphate was covalently linked to a 9-amino acridine derivative via a pentamethylene linker. Two oligo-[beta]-deoxynucleotides containing the complementary sequence in either the 5'----3' or the 3'----5' orientation were synthesized using natural [beta]-deoxynucleosides. Complex formation was investigated by absorption and fluorescence spectroscopies. No change in spectroscopic properties was detected with the anti-parallel [beta] sequence. Absorption changes were induced in the visible absorption band of the acridine derivative at 2 degrees C when the acridine-substituted oligo-[alpha]-deoxynucleotide was mixed in equimolecular amounts with the complementary [beta]-sequence in the parallel orientation. Hypochromism was observed in the UV range. The fluorescence of the acridine derivative was quenched by the guanine base present in the second position of the complementary sequence. Cooperative dissociation curves were observed and identical values of melting temperatures were obtained by absorption and fluorescence. An increase in salt concentration stabilized the complex with a delta Tm of 8 degrees C when NaCl concentration increased from 0.1 to 1 M. These results demonstrate that an oligo-[alpha]-deoxynucleotide covalently linked to an intercalating agent is able to form a double helix with an oligo-[beta]-deoxynucleotide. The two strands of this [alpha]-[beta] double helix adopt a parallel 5'----3' orientation. The acridine ring is able to intercalate between the first two base pairs on the 5'-side of the duplex structure.

Base Sequence↗
PubMed1987

Conformations of duplex structures formed by oligodeoxynucleotides covalently linked to the intercalator 2-methoxy-6-chloro-9-aminoacridine.

A family of covalent complexes between oligonucleotides and derivatives of the intercalating agent 9-amino acridine has been synthesized (Asseline, U., Thuong, N.T. and Helene, C. (1983) C.R.Acad. Sci. (Paris) 297 (III), 369-372) and studied (Lancelot, G., Asseline, U., Thuong, N.T., and Helene, C. (1985) Biochemistry 24, 2521-2529; Lancelot, G., Asseline, U., Thuong, N.T., and Helene, C. (1985) J. Biomol. Str. Dyn. 3, 913-921) with a view to understand nucleic acid-nucleic acid recognition. In order to understand the nature of interactions between the intercalator and the oligonucleotides in such complexes and the sensitivity of such interactions to the polymorphic form of the DNA, we have carried out molecular mechanics simulations on duplex deoxyoligonucleotides d(A)6.d(T)6 (A and B forms) and d(TATC).d(GATA) (B form) covalently bound to 2-methoxy-6-chloro-9-aminoacridine through a pentamethylene linker chain. Structures in which the acridine derivative is end stacked (at the 3' and 5' ends) and in which the dye is intercalated between the terminal base pairs (at both the ends) and between second and third base pairs from the 3' end are all of reasonably low energy in both A and B forms of DNA. Our studies on 3' end complexes find that in the B form, intercalation of the dye between the second and third base pairs is preferred over the other two modes of binding, while in the A form, intercalation between the terminal base pairs is preferred. In the 5' end A and B form complexes, outside stacking and intercalation between the terminal base pairs are preferred, respectively. Our calculations suggest the possibility that the presence of the dye attached covalently to the DNA can induce conformational transitions in the DNA. For example, intercalation of the dye two base pairs from the end could induce an A----B transition.

Aminoacridines↗
PubMed1985

Platelet cytosolic free calcium concentration in primary hypertension.

Cytosolic free Ca2+ ([Ca2+]i) concentrations were measured in platelets from hypertensive and normotensive man and rat with the fluorescent indicator Quin-2/AM, taking into account the signal of the free chelator. In the absence of added external Ca2+, no difference in [Ca2+]i was observed between platelets of hypertensive patients and those of normotensive subjects or between platelets of spontaneously hypertensive rats (SHR; Okamoto-Aoki strain) and those of normotensive Wistar-Kyoto (WKY) rats. In the presence of 0.5-1 mmol/l external Ca2+, [Ca2+]i was higher in patients with essential hypertension than in their normotensive controls (250 +/- 14 versus 198 +/- 10 nmol/l; n = 30 and 36, P < 0.01). In SHR, platelet [Ca2+]i was higher than in WKY rats but did not change with age and blood pressure. Removal of external K+ or addition of 10(-4) mol/l ouabain were used to inhibit the Na+K(+)-pump. Whereas an increase in [Ca2+]i was observed in platelets from normotensives in the absence of external K+ (273 +/- 29 versus 197 +/- 9 nmol/l; n = 6; P < 0.05), no significant change in [Ca2+]i was observed after ouabain treatment (220 +/- 2 versus 203 +/- 22 nmol/l, n = 8). These results suggest that primary hypertension is accompanied by a disequilibrium between cellular Ca2+ influx, storage and extrusion. Such a characteristic, if present in other excitable cells and in particular in vascular smooth muscle cells, may play a major role in the increase in peripheral resistance. However, the relationship between Na(+)-pump inhibition and the rise in the intracellular calcium remains unclear.

Adult↗
PubMed1985

[Elevation of intraplatelet free Ca2+ in primary hypertension in man and the rat].

Cytosolic free Ca2+ concentrations were measured in platelets from hypertensive and normotensive humans and rats with the use of the fluorescent indicator Quin-2/AM. Without external Ca2+ added, no difference was observed between platelets of hypertensive patients and those normotensive subjects or between platelets of spontaneously hypertensive rats and those of the normotensive Wistar Kyoto and regular Wistar rats. In the presence of 0.5-1 mM external Ca2+, the cytosolic free Ca2+ concentrations were higher both in patients with essential hypertension and rats with genetic hypertension than in their respective normotensive controls. These results suggest that primary hypertension is accompanied by a disequilibrium between cellular Ca2+ influx, storage and extrusion. Such a characteristic if present in other excitable cells and in particular in vascular smooth muscle cells may play a major role in the rise of peripheral resistances.

Adult↗
PubMed1985

Platelet cytosolic free calcium concentration in essential hypertension.

Intraplatelet free [Ca2+] was studied in human primary hypertension using the fluorescent indicator Quin-2. The cytosolic free [Ca2+] was observed to increase in human platelets as a function of the external [Ca2+]. In the absence of added external calcium, no difference in [Ca2+]i was observed between platelets of hypertensive patients and normotensive subjects (101 +/- 5 nM vs 89 +/- 5 nM, n = 27 and 33). In the presence of 1 nM Ca2+ in the external medium, [Ca2+]i was significantly higher in hypertensive patients than those of normotensive subjects (254 +/- 16 nM vs 202 +/- 10 nM, n = 27 and 34, p less than 0.01). Since an increase in cytosolic free Ca2+ concentration has been proposed to stem from the enhanced intracellular Na+ concentration due to the Na+ pump inhibition, the effects on [Ca2+]i of ouabain and removal of external K+ ions were investigated. No effect of 10(-4)M ouabain was observed whereas [Ca2+]i rose from 198 +/- 8 to 273 +/- 29 (n = 6) in absence of external K+ ions. The increase of intracellular free [Ca2+] in platelets of essential hypertensives observed only in the presence of external Ca2+ thus reflects a structural membrane alteration leading to an increased influx and/or a limited extrusion capacity. Its relationship with cell Na+ handling remains unclear.

Adult↗
PubMed1984

Oligodeoxynucleotides covalently linked to intercalating dyes as base sequence-specific ligands. Influence of dye attachment site.

New molecules with high and specific affinity for nucleic acid base sequences have been synthesized. They involve an oligodeoxynucleotide covalently attached to an intercalating dye. Visible absorption spectroscopy and fluorescence have been used to investigate the binding of poly(rA) to octadeoxythymidylates substituted by a 9-aminoacridine derivative in different positions along the oligonucleotide chain. The 9-amino group of the acridine dye was linked through a polymethylene bridge to the 3'-phosphate, the 5'-phosphate, the fourth internucleotidic phosphate or to both the 3'- and 5'-phosphates. Different interactions of the acridine dye were exhibited by these different substituted oligodeoxynucleotides when they bind to poly(rA). The interaction was shown to be specific for adenine-containing polynucleotides. The stability of these complexes was compared with that of oligodeoxynucleotides substituted by an alkyl group on the 3'-phosphate. The increase in stability due to the presence of the intercalating dye has been determined from the comparison of melting temperatures. These results are discussed with respect to the strategy of synthesis of a new class of molecules with high affinity and high specificity for nucleic acid base sequences.

Aminacrine↗
PubMed1984

Further analysis of cell membrane changes in genetic hypertension in rats by diphenylhexatriene fluorescence polarization.

Fluorescence polarization of diphenylhexatriene embedded in membranes was used as an index of 'microviscosity' in platelets and erythrocyte ghosts of spontaneously hypertensive rats of the Okamoto-Aoki strain (SHR), Wistar-Kyoto strain (WKY) and of the hypertension-prone and -resistant Sabra strains (SBH and SBN ), and the original Sabra strain (SB). Microviscosity was increased both in erythrocyte ghosts and platelet membranes of male but not female SHR rats compared with WKY rats and in hypertension-prone Sabra rats compared with the original Sabra rats. Acute and chronic salt loading increased the microviscosity of platelet membranes in all strains of rats but had no effect on the erythrocyte membranes. Microviscosities of vesicles made of lipids extracted from SHR and WKY erythrocyte ghosts were similar. This supports the hypothesis that membrane proteins play a major role in the differences in microviscosity observed in SHR rats.

Animals↗
PubMed1984

Nucleic acid-binding molecules with high affinity and base sequence specificity: intercalating agents covalently linked to oligodeoxynucleotides.

Oligodeoxyribonucleotides covalently linked to an intercalating agent via a polymethylene linker were synthesized. Oligothymidylates attached to an acridine dye (Acr) through the 3'-phosphate group [(Tp)n(CH2) mAcr ] specifically interact with the complementary sequence. The interaction is strongly stabilized by the intercalating agent. By using absorption and fluorescence spectroscopies, it is shown that complex formation between (Tp)n(CH2) mAcr and poly(rA) involves the formation of n A X T base pairs, where n is the number of thymines in the oligonucleotide. The acridine ring intercalates between A X T base pairs. Fluorescence excitation spectra reveal the existence of two environments for the acridine ring, whose relative contributions depend on the linker length (m). The binding of (Tp)4(CH2) mAcr to poly(rA) is analyzed in terms of site binding and cooperative interactions between oligonucleotides along the polynucleotide lattice. Thermodynamic parameters show that the covalent attachment of the acridine ring strongly stabilizes the binding of the oligonucleotide to its complementary sequence. The stabilization depends on the linker length; the compound with m = 5 gives a more stable complex than that with m = 3. These results open the way to the synthesis of a family of molecules exhibiting both high-affinity and high-specificity for a nucleic acid base sequence.

Acridines↗
PubMed1984

Cell membrane changes after in vivo acute Na+ load in normotensive and spontaneously hypertensive rats.

Our previous observation of a greater increase in erythrocyte Na+ in SHR than in WKY after an acute Na+ load may result either from a genetic membrane property or from a specific plasma influence. In order to elucidate this question, membrane characteristics were compared with or without an acute Na+ load. Na+ transport was measured in Ringer and in plasma on Na+ enriched and K+ depleted red cells. Platelet microviscosity was measured as an index of membrane structural changes. After acute Na+ load a similar reduction of net Na+ extrusion and of K+ influx was observed in both strains. This indicates an inhibition of the Na+,K+-pump. Platelet microviscosity was similarly increased in SHR and WKY. Thus an acute Na+ load induced alterations of membrane properties in both SHR and WKY. The higher erythrocyte Na+ content in SHR stems rather from their intrinsic membrane properties than from a plasma factor.

Animals↗
PubMed1983

Excitation energy transfer from tryptophan residues of peptides and intrinsic proteins to diphenylhexatriene in phospholipid vesicles and biological membranes.

An efficient excitation energy transfer from tryptophan residues of intrinsic membrane proteins to an extrinsic fluorescent probe (diphenylhexatriene) has been demonstrated in rat erythrocyte ghosts. To correlate this transfer with the localization of the probe, a model system has been investigated. It consists of peptides containing lysine and tryptophan residues bound to negatively charged phosphatidylserine vesicles. Absorption and fluorescence spectroscopies were used to follow peptide binding and diphenylhexatriene incorporation. Peptide binding is accompanied by a blue shift of the tryptophan fluorescence together with an increase of the quantum yield and of the fluorescence decay time. An experimental Föster critical distance value of 4.0 nm was found for energy transfer from tryptophan residues of peptides to diphenylhexatriene which approaches the range of calculated values (3.1-3.7 nm) using a two-dimensional model. These results demonstrate that efficient energy transfer can occur from tryptophan residues of intrinsic proteins to diphenylhexatriene without any interaction between diphenylhexatriene and proteins in biological membranes.

Animals↗
PubMed1982

Diffuse structural alterations in cell membranes of spontaneously hypertensive rats.

Plasma membranes from heart, nerve endings, and liver were compared in 3-week-old male spontaneously hypertensive rats from the Okamoto substrain (SHR) and normotensive Wistar/Kyoto control rats (WKY) [systolic blood pressure 105 +/- 4 and 95 +/- 4 mm Hg, respectively (1 mm Hg = 133 Pa)] according to two criteria: calcium binding at physiological intracellular concentrations and polarization of an embedded fluorescent probe, 1,6-diphenyl-1,3,5-hexatriene. Whatever the tissue of origin, the density of high-affinity calcium binding sites was lower in SHR than in WKY plasma membranes, and the polarization of diphenylhexatriene fluorescence was constantly higher in SHR than in WKY membranes. These membrane abnormalities are similar to those previously described in the erythrocyte membrane from SHR. The presence of diffuse structural alterations in cellular membrane from young spontaneously hypertensive rats when blood pressure is still in the normotensive range suggests a genetic origin. Such inherited abnormalities may by themselves participate in the rise in blood pressure.

Animals↗
PubMed1980

Conformation of two homologous neurotoxins. Fluorescence and circular dichroism studies.

Two homologous short neurotoxins isolated from snake venoms (Laticauda semifasciata erabutoxin b and Naja nigricollis toxin alpha) have been studied by means of fluorescence spectroscopy and in aqueous solution at various pH values. In parallel experiments, the stability of toxin conformations was analyzed on the basis of ultraviolet circular dichroism. Total luminescence spectra (77 K) were recorded for both toxins in neutral and alkaline solutions. The data obtained indicate that, at neutral pH, the fluorescence emission is only due to the single and invariant tryptophan (29). From a comparative study with erabutoxin a, which differs from erabutoxin b by a single residue, it is unambiguously shown that the protonation of His-26 of erabutoxin b is responsible for a decrease of Trp-29 fluorescence. Also, on the basis of available X-ray data it is proposed that the protonation or deprotonation of the following titrable groups is responsible for an alteration of Trp-29 fluorescence. These are Asp-31 (pK congruent to 4) and Lys-27 (pK = 9.6) for both toxins and Lys-26 (pK congruent to 9.6) for toxin alpha. No tyrosinate emission can be observed at neutral pH and 77 K. Excitation spectra of toxin alpha revealed that 50% of the light absorbed by Tyr-25 in water is transferred to Trp-29. From the energy transfer measurements, the distance separating these two aromatic chromophores in the native toxin was estimated to be 13 A. A similar experiment was made for toxin alpha dissolved in trifluorethanol. The data indicate that the distance separating the two aromatic side chains does not depend greatly on the nature of the solvent.

Animals↗
PubMed1979

The role of tyrosine in the association of proteins and nucleic acids. Specific recognition of single-stranded nucleic acids by tyrosine-containing peptides.

Oligopeptides containing tyrosyl, lysyl, and alanyl residues bind to polynucleotides and nucleic acids as shown by proton magnetic resonance, fluorescence spectroscopy, and difference absorption spectroscopy. Proton magnetic resonance data indicate that stacking of tyrosyl residues with nucleic acid bases takes place only in single-stranded structures (such as poly(A) or denatured DNA). Stacking interactions lead to a quenching of tyrosine fluorescence. However, the tyrosyl fluorescence of the peptides is quenched in their complexes with both single-stranded and double-stranded nucleic acids. A comparison of the behavior of homologous peptides containing Tyr, methoxytyrosine, and Phe leads to the conclusion that hydrogen bonding of tyrosine with bases or phosphates is not involved in the investigated complexes. An energy transfer mechanism from tyrosine to nucleic acid bases is proposed to account for fluorescence quenching in oligopeptide complexes with double-stranded DNAs. Due to the specificity of its stacking interaction for single-stranded nucleic acid structures, tyrosine might be involved through such interactions in the selective recognition of single strands by proteins.

Alanine↗