Search PubMed⌕ Search

Biomedical subjects

E Lacroix

Publications and source records attributed to E Lacroix.

At least 19 recordsLinked to original sources

Computer-assisted re-design of spectrin SH3 residue clusters.

We have developed a protein design computer program, called Perla, which performs searches in sequence space to uncover optimal amino acid sequences for desired protein three-dimensional structures. Optimal sequences are localised at the minima of a sequence-structure energy landscape defined using a complex scoring function (an all-atom molecular mechanics force field plus statistical terms including entropy and solvation) measured with respect to a reference state simulating a denatured protein. Sequence choices eventually optimise side chain packing, secondary structure propensities, and hydrogen bonding and electrostatics interactions. Perla was used to re-design clusters of residues of the SH3 domain of alpha-spectrin. Several mutant proteins were produced and characterised. Some of our designed proteins have significantly higher stabilities (stability enhancements about 0.25, 0.70 and 1.0 kcal mol(-1)) than the wild-type protein. These successful protein re-designs, and similar examples found in the literature, establish the quality of the structure-based computational approach to protein design.

Algorithms↗

Computer-aided design of beta-sheet peptides.

The design of beta-sheet proteins is still a challenge in the field of de novo protein design. Here, we have tested the validity of automatic design methods to create and/or improve beta-sheet peptides and proteins. We chose Betanova, a three-stranded beta-sheet peptide, as target system, and, as an automatic design tool, a protein design algorithm called PERLA (protein engineering rotamer library algorithm). PERLA was used to define both stabilising and destabilising single- and multiple-residue mutations of Betanova. Conformational analysis by NMR spectroscopy and far-UV circular dichroism (CD) allowed us to evaluate population differences among the set of designed peptides. Some of the new mutants are approximately 1 kcal/mol more stable than the wild-type peptide. Comparison of the scale of predicted and observed stabilities demonstrates that they are in good agreement for most peptides studied. Our results show that automatic design algorithms can be successfully applied to the design of beta-sheet peptides.

Algorithms↗

Multifunctionalized alpha,beta-cyclopentenones from C-2 and C-4-ulopyranosyl compounds: a stereospecific rearrangement initiated by base.

Base treatment of O-benzyl protected C-2- or C-4-ulopyranosyl compounds (4 alpha, 4 beta, and 11) by either 10% Et(3)N or 1% K(2)CO(3) in MeOH initiated a beta elimination to afford alpha,beta-unsaturated C-ulopyranosyl compounds (5 alpha, 5 beta, and 12), which further rearranged in a stereocontrolled manner to multifuctionalized alpha,beta-cyclopentenones (6 and 14) in 70-80% yield. Both C-alpha- and C-beta-2-ulosides (5 alpha and 5 beta) produced the same cyclopentenone 6, indicating that a 1,2-enolate is formed prior to the cleavage of the C-5--O bond. Because 6 is racemic, it was probably formed by the intramolecular cycloaldolization of two equally populated enantiomeric intermediates. When treated with 90% Et(3)N in MeOH, 5 alpha yielded almost exclusively 15 (isomer of 6), which was formed by a migration of the double bond in 5 alpha during the previously described rearrangement. Thus either 6 or 15 was the major product, depending on the base used.

Cyclopentanes↗

Computational estimation of specific side chain interaction energies in alpha helices.

We have used a structure energy-based computer program developed for protein design, Perla, to provide theoretical estimates of all specific side chain-side chain interaction energies occurring in alpha helices. The computed side chain-side chain interaction energies were used as substitutes for the corresponding values used by the helix/coil transition algorithm, AGADIR. Predictions of peptide helical contents were nearly as successful as those obtained with the originally calibrated set of parameters; a correlation to experimentally observed alpha-helical populations of 0.91 proved that our theoretical estimates are reasonably correct for amino acid pairs that are frequent in our database of peptides. Furthermore, we have determined experimentally the previously uncharacterized interaction energies for Lys-Ile, Thr-Ile, and Phe-Ile amino acid pairs at i,i + 4 positions. The experimental values compare favorably with the computed theoretical estimates. Importantly, the computed values for Thr-Ile and Phe-Ile interactions are better than the energies based on chemical similarity, whereas for Lys-Ile they are similar. Thus, computational techniques can be used to provide precise energies for amino acid pairwise interactions, a fact that supports the development of structure energy-based computational tools for structure predictions and sequence design.

Circular Dichroism↗

Towards understanding a molecular switch mechanism: thermodynamic and crystallographic studies of the signal transduction protein CheY.

The signal transduction protein CheY displays an alpha/beta-parallel polypeptide folding, including a highly unstable helix alpha4 and a strongly charged active site. Helix alpha4 has been shown to adopt various positions and conformations in different crystal structures, suggesting that it is a mobile segment. Furthermore, the instability of this helix is believed to have functional significance because it is involved in protein-protein contacts with the transmitter protein kinase CheA, the target protein FliM and the phosphatase CheZ. The active site of CheY comprises a cluster of three aspartic acid residues and a lysine residue, all of which participate in the binding of the Mg(2+) needed for the protein activation. Two steps were followed to study the activation mechanism of CheY upon phosphorylation: first, we independently substituted the three aspartic acid residues in the active site with alanine; second, several mutations were designed in helix alpha 4, both to increase its level of stability and to improve its packing against the protein core. The structural and thermodynamic analysis of these mutant proteins provides further evidence of the connection between the active-site area and helix alpha 4, and helps to understand how small movements at the active site are transmitted and amplified to the protein surface.

Amino Acid Substitution↗

The design of linear peptides that fold as monomeric beta-sheet structures.

Current knowledge about the determinants of beta-sheet formation has been notably improved by the structural and kinetic analysis of model peptides, by mutagenesis experiments in proteins and by the statistical analysis of the protein structure database (Protein Data Bank; PDB). In the past year, several peptides comprising natural and non-natural amino acids have been designed to fold as monomeric three-stranded beta-sheets. In all these cases, the design strategy has involved both the statistical analysis of the protein structure database and empirical information obtained in model beta-hairpin systems and in proteins. Only in one case was rotamer analysis performed to check for the compatibility of the sidechain packing. It is foreseeable that, in future designs, algorithms exploring the sequence and conformational space will be employed. For the design of small proteins (less than 30 amino acids), questions remain about the demonstration of two-state behavior, the formation of a well-defined network of mainchain hydrogen bonds and the quantification of the structured populations.

Crystallography, X-Ray↗

[Comparative study of the ideas about causes, disease mechanisms and therapies of the plague on the basis of the plague treatises of the Medical Faculty of Paris (1348-1349), of Joannes de Vesalia (after 1454) and of Thomas Montanus (1669)].

The comparison between the consilium of the Faculty of Paris (14th century), and the treatises by Joannes de Vesalia (15th century) and by Thomas Montanus (17th century) shows that the concepts with regard to the causes and the mechanisms of and the proposed preventive and curative measures against the plague did in essence not change for over three centuries. In fact this is not very surprising, since the development of modern physiology and physiopathology only started gradually in the second half of the 19th century. Furthermore, the plague bacillus has only been identified in 1894. However, the preventive and curative measures against plague that are proposed in the three treatises, do not result from an uncontrolled imagination. Most of these measures have a rational basis and are the result of--although erroneous--concepts with regard to the causes and mechanisms of the disease.

Education, Medical↗

Elucidating the folding problem of alpha-helices: local motifs, long-range electrostatics, ionic-strength dependence and prediction of NMR parameters.

The information about the conformational behavior of monomeric helical peptides in solution, as well as the alpha-helix stability in proteins, has been previously utilized to derive a database with the energy contributions for various interactions taking place in an alpha-helix: intrinsic helical propensities, side-chain-side-chain interactions, main-chain-main-chain hydrogen bonds, and capping effects. This database was implemented in an algorithm based on the helix/coil transition theory (AGADIR). Here, we have modified this algorithm to include previously described local motifs: hydrophobic staple, Schellman motif and Pro-capping motif, new variants of these, and newly described side-chain-side-chain interactions. Based on recent experimental data we have introduced a position dependence of the helical propensities for some of the 20 amino acid residues. A new electrostatic model that takes into consideration all electrostatic interactions up to 12 residues in distance in the helix and random-coil conformations, as well as the effect of ionic strength, has been implemented. We have synthesized and analyzed several peptides, and used data from peptides already analysed by other groups, to test the validity of our electrostatic model. The modified algorithm predicts, with an overall standard deviation value of 6.6 (maximum helix is 100%), the helical, content of 778 peptides of which 223 correspond to wild-type and modified protein fragments. To improve the prediction potential of the algorithm and to have a direct comparison with nuclear magnetic resonance data, the algorithm now predicts the conformational shift of the CalphaH protons, 13Calpha and 3JalphaN values. We have found that for those peptides correctly predicted from the point of view of circular dichroism, the prediction of the NMR parameters is very good.

Algorithms↗

Reading protein sequences backwards.

BACKGROUND: Reading a protein sequence backwards provides a new polypeptide that does not align with its parent sequence. The foldability of this new sequence is questionable. On one hand, structure prediction at low resolution using lattice simulations for such a protein provided a model close to the native parent fold or to a topological mirror image of it. On the other hand, there is no experimental evidence yet to tell whether such a retro protein folds (and to which structure) or not. RESULTS: In this work, we have analysed the possibility of a retro protein folding in two different ways. First, we modelled the retro sequence of the alpha-spectrin SH3 domain through distance geometry and molecular dynamics. This contradicted the plausibility of a mirror image of the native domain, whereas basic considerations opposed the likelihood of the native fold. Second, we obtained experimental evidence that the retro sequences of the SH3 domain, as well as the B domain of Staphylococcal protein A and the B1 domain of Streptococcal protein G, are unfolded proteins, even though some propensities for the formation of secondary structures might remain. CONCLUSIONS: Retro proteins are no more similar to their parent sequences than any random sequence despite their common hydrophobic/hydrophilic pattern, global amino acid composition and possible tertiary contacts. Although simple folding models contribute to our global understanding of protein folding, they cannot yet be used to predict the structure of new proteins.

Amino Acid Sequence↗

Amide hydrogen exchange and internal dynamics in the chemotactic protein CheY from Escherichia coli.

The backbone internal dynamics of the wild-type 129 amino acid alpha/beta parallel protein CheY and its double mutant F14N/P110G are analysed here by the hydrogen-exchange method. The F14N mutation is known to stabilise the protein and to accelerate refolding while P110G is destabilising and accelerates unfolding. We first assigned and characterised the double mutant by nuclear magnetic resonance (NMR), to try and discover any possible conformational change induced by the two mutations. The main difference between the two proteins is a favourable N-capping interaction of the newly introduced Asn14 side-chain at the beginning of the first alpha-helix (alpha-helix A). Second, we have measured the exchange rates in the wild-type and mutant CheY. In the first case the observed protection factors are slightly dispersed around an average value. According to their distribution in the structure, protein stability is highest on one face of the central beta-sheet, in the surroundings of the main hydrophobic core formed by side-chains of residues in beta-strands I, II and III and helices A and E. The mutations in the double mutant protein affect two distinct subdomains differently (from beta-strand I to III and from alpha-helix C to the end). In the second subdomain the number of protected protons is reduced with respect to those in the wild-type. This differential behaviour can be explained by a selective decrease in stability of the second folding subdomain produced by the P110G mutation and the opposite effect in the first subdomain, produced by the F14N mutation. alpha-Helix A, which is involved together with beta-strands I and III in the folding nucleus of CheY, shows the largest protection factors in both proteins.

Amides↗

[From empirical 'dietetics' to rational dietetics].

From the outset, the terms 'dietetics' and 'diet' applied in their general acceptance to all the measures, whether or not alimentary, that can provide optimal living conditions for both the healthy and the diseased. In that meaning they are synonymous with hygiene or way of life. In their more restricted acceptance, which will be used henceforth, the terms apply to the choice of food and beverages which promotes or restores health of normal and sick people. In its most restricted meaning, the term 'diet' is confined to the nutrition of the sick, consisting either in a more or less drastic limitation of food intake, or in the prescription of specific foods. Due to the absence of scientific knowledge concerning the processes of life in general, and in particular concerning the physiology of the digestive system and the metabolism, the 'dietetic' rules and principles were governed until the middle of the nineteenth century by empiricism with a strong background of intuition, tradition, magic and religion. In his continuous struggle for life, man has been confronted with the dramatic consequences of the ingestion of toxic and spoiled food. Nausea, vomiting, diarrhea, cramps and eventually death have soon been linked with the ingestion of particular food, although the symptoms were attributed to supernatural forces. Intuitively and later by laws and prohibition, the use of certain foods was to be avoided or was forbidden. Moreover, with the development of cultures and civilizations food and food intake have acquired -besides their vital necessity-social and often magic-religious values which sometimes resulted in food taboos. In most civilizations these taboos have particulars in common: they are mainly directed against food of animal origin, more against meat than fish and particularly against red meat, which is being considered as symbol of strength and power and as endowed with exciting and stimulatory properties. The meat taboos are selective for some animals or parts of an animal, but what is allowed in some cultures is forbidden in others. More or less severe and long lasting abstinence from food or fasting was also self-inflicted or imposed to individuals or whole populations by religion, sometimes seemingly on hygienic grounds but in most cases without any logic reason. Since the earliest time, diets take-together with other dietetic measures-a prevailing place in the treatment of the sick and sickness. Most alimentary prescriptions are based on a more or less drastic restriction of food and - particularly in the seventeenth century with the iatrochemical school - also of fluids. The restrictions were not only quantitative, but also qualitative with restriction of meat consumption-particularly of red meat-and the prescription of more or less diluted decoctions of barley (infusions) or broth. The numerous works on 'dietetics' published-thanks to the development of printing-from the seventeenth on through the nineteenth century concern mainly the description of foodstuffs and beverages available at the time, and of the ways of preparing them. They also provide indications concerning the way these foodstuffs can improve health and/or list which of them can be prescribed or must be avoided in the treatment of the sick and of specific diseases. Some of these 'dietetical' prescriptions and maxims seem perfectly valid from a scientific and therapeutical point of view, - at least according to our actual knowledge - however, many are totally ineffective or are ridiculous and even dangerous. Except for the "dietetical" measures imposed by public hygiene or by religion to everyone, most of the dieteticotherapeutical prescriptions (food restrictions, clysters, purgation, bleeding) involved only the small fraction of the population that could afford medical assistance. The common people - the majority - could afford neither medical help, nor food abuse - except for an occasional feast - and had only access to a limited quantity and choi

Diet↗

Influence of converting enzyme inhibition on isoflurane-induced hypotension for cerebral aneurysm surgery.

Twenty-five patients (aged 18 to 72 years), who recovered after the first bleed from a cerebral aneurysm, were operated on under neuroleptanaesthesia. Isoflurane was added to induce hypotension. It was found that the required hypotension (51 (SEM 1) mmHg) could be obtained and maintained at much lower isoflurane concentrations (less than 1%) after blockade of the angiotensin converting enzyme activity by enalaprilat (2.5 mg i.v.) than without such inhibition. During the hypotension which lasted 78 (SEM 10) min, only minor adjustments of the isoflurane concentration (0.70 (0.04%) were needed. The desired level of hypotension was obtained with preservation of the cardiac output and without tachycardia. No resistance to the blood pressure lowering effect of isoflurane was observed. On recovery from anaesthesia, a small increase of blood pressure above control values was seen in 16 patients and was easily reversed by small doses of clonidine (mean total dose: 220 (61) micrograms). The operative conditions were excellent and the postoperative recovery was uneventful and complete in 23 patients.

Adolescent↗

Idazoxan inhibits hyperprolactinaemia in ovariectomized estrogen-treated rats.

The alpha 2-antagonist idazoxan (IDZ) has previously been shown to inhibit hyperprolactinaemia triggered by various stimuli such as lactation, stress, serotonergic agents and morphine (Preziosi, Martire, Navarra, Pistritto and Vacca 1989; Krulich, Jurcovicova and Le 1989). In this study, we investigated the PRL-lowering activity of IDZ in ovariectomized estrogen-treated (OET) rats; since a PRL surge usually occurs in normal cycling rats on the day of proestrus, the effect of IDZ on pulsatile PRL release in intact female rats was also studied. IDZ significantly lowered plasma PRL levels in OET rats; no elevated PRL values were observed in normal cycling rats, indicating that IDZ might inhibit PRL surges in these animals. It is concluded that IDZ is an effective PRL-lowering agent in a number of physiological and pharmacological hyperprolactinaemic models.

Animals↗

Alpha 2-adrenoceptor-mediated inhibition of prolactin release in suckling- or fenfluramine-induced hyperprolactinemia.

It has recently been shown that the specific and selective alpha 2-antagonist idazoxan (IDZ) displays prolactin-lowering activity on hyperprolactinemia induced in the rat either by suckling or serotonergic drugs. In an attempt better to understand the role of alpha 2-adrenoceptors under the above conditions, experiments were carried out to compare the effects of IDZ with that of the classic alpha 2-antagonist yohimbine (YOH), and also of the alpha 2-agonists clonidine (CLO) and B-HT 920, on prolactin (PRL) release during lactation and in hyperprolactinemia induced in male rats by the serotonergic drug fenfluramine (FEN). In lactating rats, both alpha 2-agonists decreased PRL release; this effect was enhanced by prior separation of the animals from their pups for several hours. A decrease of plasma PRL levels was also induced by IDZ but not by YOH, which tended further to increase hyperprolactinemia. In male rats treated with FEN, IDZ and CLO, a significant decrease of plasma PRL was produced, but YOH further enhanced PRL secretion. It is concluded that the alpha 2-agonists tested and also the alpha 2-antagonist IDZ display a unique inhibitory activity on PRL release during suckling or serotonergic-induced hyperprolactinemia.

Adrenergic alpha-Agonists↗

Regulation of the pituitary 5 alpha-reductase activity by gonadotropin releasing hormone and testosterone in the adult male rat.

Intact or castrated adult male rats were treated for nine days with GnRH (10 micrograms/day), the synthetic GnRH goserelin (100 micrograms/day) or the GnRH-antagonist Org 30276 (250 or 500 micrograms/day). In some series, 1 mg testosterone propionate was administered alone, or in combination with goserelin or Org 30276. The in vitro metabolism of [1 alpha,2 alpha-3H]testosterone by pituitary and hypothalamic homogenates was investigated in combination with the estimation of plasma concentrations of testosterone and gonadotropins. No qualitative or quantitative differences were observed in hypothalamic testosterone metabolism or in the pituitary 17 beta-hydroxysteroid dehydrogenase activity. Testosterone administration to intact male rats decreased the pituitary 5 alpha-reductase activity and LH, while administered to castrated rats, it was able to suppress totally the castration-induced increase of the 5 alpha-reductase activity and of the gonadotropin secretion. The drastic decrease of the plasma levels of testosterone, observed after a prolonged treatment with GnRH, goserelin or Org 30276 was not accompanied by an increased pituitary 5 alpha-reductase activity. Injected to castrated rats, it was observed that the castration-induced increase of the pituitary 5 alpha-reductase was further stimulated by GnRH, totally suppressed by goserelin and partially suppressed by Org 30276. Concomitant administration of goserelin or Org 30276 and testosterone propionate to castrated rats resulted in a further decrease of the pituitary 5 alpha-reductase activity, compared to the castrated, GnRH-analogue treated rats. These data indicate that the pituitary 5 alpha-reductase enzyme system is controlled by both direct steroidal and indirect GnRH-mediated mechanisms.

3-Oxo-5-alpha-Steroid 4-Dehydrogenase↗

Effect of tamoxifen on the activity of enzymes of testicular steroidogenesis.

Testicular homogenates of tamoxifen-treated rats were incubated with labeled steroid precursors (progesterone, 17 alpha-hydroxyprogesterone, dehydroepiandrosterone, androstenedione or testosterone) in order to study the effect of tamoxifen on testicular steroidogenesis. The results indicate that a 9 day treatment with a daily dose of 1 mg tamoxifen produces a reduction of the synthesis of testosterone. Inhibition of the 17 alpha-hydroxylase and C17,20-desmolase enzyme systems was observed together with an increased 20 alpha-hydroxysteroid dehydrogenase activity.

20-Hydroxysteroid Dehydrogenases↗

The effects of neonatal androgenization of male rats on testosterone metabolism by the hypothalamus-pituitary-gonadal axis.

Male rats were androgenized on the third postnatal day by a single injection of 1 mg testosterone propionate. The in vitro metabolism of [4-14C]testosterone by pituitary and hypothalamus homogenates was investigated at the age of 90 days. The pituitary and hypothalamus homogenates from control and neonatally androgenized animals converted [4-14C]testosterone to the same metabolites, mainly 5 alpha-reduced derivatives; the quantitative yield of 5 alpha-reduced metabolites was much higher in the pituitary homogenates of androgenized rats. The hypothalamic homogenates showed no differences. In the androgenized rats a very significant increase of the plasma FSH levels was measured while the LH levels were also augmented. The plasma levels of testosterone were not different from the values in control rats, notwithstanding a 25% reduction in testes weight. The present experiments appear to indicate that the neonatal androgenization results in an accentuation of the sexual dimorphism which normally exists in the pituitary of adult rats for the 5 alpha-reductase activity.

Androgens↗

Influence of a prolonged tamoxifen administration on steroidogenesis by incubated rat testes.

Tamoxifen was administered i.m. for 9 days to adult male rats in a daily dose of 100 micrograms or 1 mg. The treatment resulted in a significant reduction of the plasma levels of testosterone and LH, without modification of the plasma levels of FSH and of the testes weight. Upon incubation, the testes from the tamoxifen-treated rats produced less testosterone and 7 alpha-hydroxytestosterone, but metabolized [4-14C]testosterone in the same way as the control animals. Small doses of hCG (0.5 i.u. for 9 days) were unable to modify the tamoxifen effect on the testicular function, while tamoxifen significantly inhibited the increase of the plasma levels of testosterone induced by the administration of moderate doses of hCG (1.5 i.u. or 2.5 i.u. for 9 days) to hypophysectomized rats. Tamoxifen treatment, however, did not modify significantly the reactivity of the testes towards high doses of hCG (10 i.u.), administered either 2 h before sacrifice or for 9 days. It is concluded that a prolonged administration of tamoxifen in the rat has, besides an indirect effect resulting from a decrease of the LH levels, a direct inhibitory influence on the testicular testosterone formation, which can be reversed by high doses of hCG.

Androgens↗