Étude des mécanismes d’extraction lipidique par le peptide mélittine et la protéine BSP1

Les peptides et protéines extracteurs de lipides (PEL) se lient aux membranes lipidiques puis en extraient des lipides en formant de plus petits auto-assemblages, un phénomène qui peut aller jusqu'à la fragmentation des membranes. Dans la nature, cette extraction se produit sur une gamme de cellules et entraîne des conséquences variées, comme la modification de la composition de la membrane et la mort de la cellule. Cette thèse se penche sur l’extraction lipidique, ou fragmentation, induite par le peptide mélittine et la protéine Binder-of-SPerm 1 (BSP1) sur des membranes lipidiques modèles. Pour ce faire, des liposomes de différentes compositions sont préparés et incubés avec la mélittine ou la BSP1. L'association aux membranes est déterminée par la fluorescence intrinsèque des PEL, tandis que l'extraction est caractérisée par une plateforme analytique combinant des tests colorimétriques et des analyses en chromatographie en phase liquide et spectrométrie de masse (LCMS). La mélittine fait partie des peptides antimicrobiens cationiques, un groupe de PEL très répandu chez les organismes vivants. Ces peptides sont intéressants du point du vue médical étant donné leur mode d’action qui vise directement les lipides des membranes. Plusieurs de ceux-ci agissent sur les membranes des bactéries selon le mécanisme dit « en tapis », par lequel ils s’adsorbent à leur surface, forment des pores et ultimement causent leur fragmentation. Dans cette thèse, la mélittine est utilisée comme peptide modèle afin d’étudier le mécanisme par lequel les peptides antimicrobiens cationiques fragmentent les membranes. Les résultats montrent que la fragmentation des membranes de phosphatidylcholines (PC) est réduite par une déméthylation graduelle de leur groupement ammonium. L'analyse du matériel fragmenté révèle que les PC sont préférentiellement extraites des membranes, dû à un enrichissement local en PC autour de la mélittine à l'intérieur de la membrane. De plus, un analogue de la mélittine, dont la majorité des résidus cationiques sont neutralisés, est utilisé pour évaluer le rôle du caractère cationique de la mélittine native. La neutralisation augmente l'affinité du peptide pour les membranes neutres et anioniques, réduit la fragmentation des membranes neutres et augmente la fragmentation des membranes anioniques. Malgré les interactions électrostatiques entre le peptide cationique et les lipides anioniques, aucune spécificité lipidique n'est observée dans l'extraction. La BSP1 est la protéine la plus abondante du liquide séminal bovin et constitue un autre exemple de PEL naturel important. Elle se mélange aux spermatozoïdes lors de l’éjaculation et extrait des lipides de leur membrane, notamment le cholestérol et les phosphatidylcholines. Cette étape cruciale modifie la composition lipidique de la membrane du spermatozoïde, ce qui faciliterait par la suite la fécondation de l’ovule. Cependant, le contact prolongé de la protéine avec les spermatozoïdes endommagerait la semence. Cette thèse cherche donc à approfondir notre compréhension de ce délicat phénomène en étudiant le mécanisme moléculaire par lequel la protéine fragmente les membranes lipidiques. Les résultats des présents travaux permettent de proposer un mécanisme d’extraction lipidique en 3 étapes : 1) L'association à l’interface des membranes; 2) La relocalisation de l’interface vers le cœur lipidique; 3) La fragmentation des membranes. La BSP1 se lie directement à deux PC à l'interface; une quantité suffisante de PC dans les membranes est nécessaire pour permettre l'association et la fragmentation. Cette liaison spécifique ne mène généralement pas à une extraction lipidique sélective. L'impact des insaturations des chaînes lipidiques, de la présence de lysophosphatidylcholines, de phosphatidyléthanolamine, de cholestérol et de lipides anioniques est également évalué. Les présentes observations soulignent la complexe relation entre l'affinité d'un PEL pour une membrane et le niveau de fragmentation qu'il induit. L'importance de la relocalisation des PEL de l'interface vers le cœur hydrophobe des membranes pour permettre leur fragmentation est réitérée. Cette fragmentation semble s'accompagner d'une extraction lipidique préférentielle seulement lorsqu'une séparation de phase est induite au niveau de la membrane, nonobstant les interactions spécifiques PEL-lipide. Les prévalences des structures amphiphiles chez certains PEL, ainsi que de la fragmentation en auto-assemblages discoïdaux sont discutées. Finalement, le rôle des interactions électrostatiques entre les peptides antimicrobiens cationiques et les membranes bactériennes anioniques est nuancé : les résidus chargés diminueraient l'association des peptides aux membranes neutres suite à l'augmentation de leur énergie de solvatation.

Étude des mécanismes d’extraction lipidique par le peptide mélittine et la protéine BSP1

Les peptides et protéines extracteurs de lipides (PEL) se lient aux membranes lipidiques puis en extraient des lipides en formant de plus petits auto-assemblages, un phénomène qui peut aller jusqu'à la fragmentation des membranes. Dans la nature, cette extraction se produit sur une gamme de cellules et entraîne des conséquences variées, comme la modification de la composition de la membrane et la mort de la cellule. Cette thèse se penche sur l’extraction lipidique, ou fragmentation, induite par le peptide mélittine et la protéine Binder-of-SPerm 1 (BSP1) sur des membranes lipidiques modèles. Pour ce faire, des liposomes de différentes compositions sont préparés et incubés avec la mélittine ou la BSP1. L'association aux membranes est déterminée par la fluorescence intrinsèque des PEL, tandis que l'extraction est caractérisée par une plateforme analytique combinant des tests colorimétriques et des analyses en chromatographie en phase liquide et spectrométrie de masse (LCMS). La mélittine fait partie des peptides antimicrobiens cationiques, un groupe de PEL très répandu chez les organismes vivants. Ces peptides sont intéressants du point du vue médical étant donné leur mode d’action qui vise directement les lipides des membranes. Plusieurs de ceux-ci agissent sur les membranes des bactéries selon le mécanisme dit « en tapis », par lequel ils s’adsorbent à leur surface, forment des pores et ultimement causent leur fragmentation. Dans cette thèse, la mélittine est utilisée comme peptide modèle afin d’étudier le mécanisme par lequel les peptides antimicrobiens cationiques fragmentent les membranes. Les résultats montrent que la fragmentation des membranes de phosphatidylcholines (PC) est réduite par une déméthylation graduelle de leur groupement ammonium. L'analyse du matériel fragmenté révèle que les PC sont préférentiellement extraites des membranes, dû à un enrichissement local en PC autour de la mélittine à l'intérieur de la membrane. De plus, un analogue de la mélittine, dont la majorité des résidus cationiques sont neutralisés, est utilisé pour évaluer le rôle du caractère cationique de la mélittine native. La neutralisation augmente l'affinité du peptide pour les membranes neutres et anioniques, réduit la fragmentation des membranes neutres et augmente la fragmentation des membranes anioniques. Malgré les interactions électrostatiques entre le peptide cationique et les lipides anioniques, aucune spécificité lipidique n'est observée dans l'extraction. La BSP1 est la protéine la plus abondante du liquide séminal bovin et constitue un autre exemple de PEL naturel important. Elle se mélange aux spermatozoïdes lors de l’éjaculation et extrait des lipides de leur membrane, notamment le cholestérol et les phosphatidylcholines. Cette étape cruciale modifie la composition lipidique de la membrane du spermatozoïde, ce qui faciliterait par la suite la fécondation de l’ovule. Cependant, le contact prolongé de la protéine avec les spermatozoïdes endommagerait la semence. Cette thèse cherche donc à approfondir notre compréhension de ce délicat phénomène en étudiant le mécanisme moléculaire par lequel la protéine fragmente les membranes lipidiques. Les résultats des présents travaux permettent de proposer un mécanisme d’extraction lipidique en 3 étapes : 1) L'association à l’interface des membranes; 2) La relocalisation de l’interface vers le cœur lipidique; 3) La fragmentation des membranes. La BSP1 se lie directement à deux PC à l'interface; une quantité suffisante de PC dans les membranes est nécessaire pour permettre l'association et la fragmentation. Cette liaison spécifique ne mène généralement pas à une extraction lipidique sélective. L'impact des insaturations des chaînes lipidiques, de la présence de lysophosphatidylcholines, de phosphatidyléthanolamine, de cholestérol et de lipides anioniques est également évalué. Les présentes observations soulignent la complexe relation entre l'affinité d'un PEL pour une membrane et le niveau de fragmentation qu'il induit. L'importance de la relocalisation des PEL de l'interface vers le cœur hydrophobe des membranes pour permettre leur fragmentation est réitérée. Cette fragmentation semble s'accompagner d'une extraction lipidique préférentielle seulement lorsqu'une séparation de phase est induite au niveau de la membrane, nonobstant les interactions spécifiques PEL-lipide. Les prévalences des structures amphiphiles chez certains PEL, ainsi que de la fragmentation en auto-assemblages discoïdaux sont discutées. Finalement, le rôle des interactions électrostatiques entre les peptides antimicrobiens cationiques et les membranes bactériennes anioniques est nuancé : les résidus chargés diminueraient l'association des peptides aux membranes neutres suite à l'augmentation de leur énergie de solvatation.

Actions of a Proline Analogue, L-Thiazolidine-4-Carboxylic Acid (T4C), on Trypanosoma cruzi

It is well established that L-proline has several roles in the biology of trypanosomatids. In Trypanosoma cruzi, the etiological agent of Chagas' disease, this amino acid is involved in energy metabolism, differentiation processes and resistance to osmotic stress. In this study, we analyzed the effects of interfering with L-proline metabolism on the viability and on other aspects of the T. cruzi life cycle using the proline analogue L- thiazolidine-4-carboxylic acid (T4C). The growth of epimastigotes was evaluated using different concentrations of T4C in standard culture conditions and at high temperature or acidic pH. We also evaluated possible interactions of this analogue with stress conditions such as those produced by nutrient starvation and oxidative stress. T4C showed a dose-response effect on epimastigote growth (IC(50) = 0.89+/-0.02 mM at 28 degrees C), and the inhibitory effect of this analogue was synergistic (p<0.05) with temperature (0.54+/-0.01 mM at 37 degrees C). T4C significantly diminished parasite survival (p<0.05) in combination with nutrient starvation and oxidative stress conditions. Pre-incubation of the parasites with L-proline resulted in a protective effect against oxidative stress, but this was not seen in the presence of the drug. Finally, the trypomastigote bursting from infected mammalian cells was evaluated and found to be inhibited by up to 56% when cells were treated with non-toxic concentrations of T4C (between 1 and 10 mM). All these data together suggest that T4C could be an interesting therapeutic drug if combined with others that affect, for example, oxidative stress. The data also support the participation of proline metabolism in the resistance to oxidative stress.

Development and validation of a fast RP-HPLC method to determine the analogue of the thyroid hormone, 3,5,3 '-triiodothyroacetic acid (TRIAC), in polymeric nanoparticles

The objective of this work was to develop and validate a rapid Reversed-Phase High-Performance Liquid Chromatography method for the quantification of 3,5,3 '-triiodothyroacetic acid (TRIAC) in nanoparticles delivery system prepared in different polymeric matrices. Special attention was given to developing a reliable reproductive technique for the pretreatment of the samples. Chromatographic runs were performed on an Agilent 1200 Series HPLC with a RP Phenomenex (R) Gemini C18 (150 x 4, 6 mm i.d., 5 mu m) column using acetonitrile and triethylamine buffer 0.1% (TEA) (40 : 60 v/v) as a mobile phase in an isocratic elution, pH 5.6 at a flow rate of 1 ml min(-1). TRIAC was detected at a wavelength of 220 nm. The injection volume was 20 mu l and the column temperature was maintained at 35 degrees C. The validation characteristics included accuracy, precision, specificity, linearity, recovery, and robustness. The standard curve was found to have a linear relationship (r(2) - 0.9996) over the analytical range of 5-100 mu g ml(-1) . The detection and quantitation limits were 1.3 and 3.8 mu g ml(-1), respectively. The recovery and loaded TRIAC in colloidal system delivery was nearly 100% and 98%, respectively. The method was successfully applied in polycaprolactone, polyhydroxybutyrate, and polymethylmethacrylate nanoparticles.

Interaction of RTD-1, a cyclic antimicrobial defensin from Rhesus macaque leukocytes, and its open chain analogue with membrane mimics

In contrast to other mammalian defensins, rhesus theta defensin-1 (RTD-1) is composed of just 18 amino acids with the backbone cyclized through peptide bonds. Antibacterial activities of both the native cyclic peptide and a linear form were examined, showing that the cyclic form was 3-fold more active than the open chain analogue, oRTD-1, although both peptides adopt very similar structures in water. It was suggested that the additional charges at the termini of oRTD-1 are the cause for its lower antimicrobial activity. Therefore, we studied the interaction of both peptides with membrane mimics composed of zwitterionic (PC) and negatively charged (PG) phospholipids, major lipid components of erythrocyte and bacterial cell membranes, respectively. Microcalorimetry showed that RTD-1 and oRTD-1 did not affect the phase behavior of PC liposomes, while in PG liposomes both peptides induced new phase transitions above the chain melting transition of the lipid. The shape and fraction differed between both peptides, depending also on their concentration, which will be discussed in terms of their antimicrobial activity.

Dual C3a/C5a receptor agonism by a C5a C-terminal analogue decapeptide

No abstract

A cyclic peptide, C5a receptor antagonist analogue confers increased potency in a model of inflammatory bowel disease in rats

No abstract

alpha-Melanocyte Stimulating Hormone Analogue AP214 Protects Against Ischemia Induced Acute Kidney Injury in a Porcine Surgical Model

Purpose: alpha-Melanocyte stimulating hormone protects kidneys against ischemia and sepsis induced acute kidney injury in rodents. We examined the efficacy of a-melanocyte stimulating hormone analogue AP214 to protect against acute kidney injury in higher vertebrates. Materials and Methods: We performed a prospective, blinded, randomized, placebo controlled study in 26 pigs. Laparoscopic technique was used for left nephrectomy and to induce complete warm ischemia in the right kidney for 120 minutes. AP214 (200 mu g/kg intravenously) was administered daily on the day of surgery and for 5 days thereafter. Kidney function was measured for 9 days. We measured changes in serum creatinine, estimated glomerular filtration rate, serum C-reactive protein and urine interleukin-18. Results: In the placebo control and AP214 groups mean peak serum creatinine was 10.2 vs 3.92 mg/dl and the estimated glomerular filtration rate nadir was 22.9 vs 62.6 ml per minute per kg (each p = 0.001). Functional nadir occurred at 72 vs 24 hours in the control vs AP214 groups. Estimated glomerular filtration rate outcome on postoperative day 9 was 118 vs 156 ml per minute per kg in the control vs AP214 groups (p = 0.04). Conclusions: We noted a robust renoprotective effect of AP214. A similar AP214 effect may be observed in humans. Future research includes mechanistic studies in pigs and a phase II human clinical trial of AP214 in kidney transplant and partial nephrectomy populations.

Use of alprostadil, a stable prostaglandin E(1) analogue, for the attenuation of rat skeletal muscle ischemia and reperfusion injury

Aim. Some stable prostaglandin analogues such as alprostadil have been used to attenuate the deleterious effects of ischemia and reperfusion injury. The aim of this paper was to test if alprostadil can decrease the ischemia- reperfusion injury in rat skeletal muscle using muscular enzymes as markers, such as aspartate aminotransferase (AST), creatine kinase (CPK), lactate dehydrogenase (LDH); degeneration products of cell membrane-malondialdehyde (MDA) and muscle glycogen storage. Methods. Thirty male Wistar rats were used in a model of hind limb ischemia achieved by infrarenal aortic cross-clamping. The animals were randomized into three equal groups (N=10) submitted to 5 hours of ischemia followed by one hour of reperfusion. The first group (control) received continuous intravenous infusion of saline solution and the second group (preischemia, GPI) received continuous intravenous infusion of alprostadil throughout the experiment starting 20 minutes before the aortic cross-clamping. The third group, prereperfusion (GPR), received alprostadil only during the reperfusion period, with intravenous infusion being started 10 min before the clamp release. Results. There was no difference in CPK, LDH, AST or tissue glycogen values between groups. However, a significant elevation in MDA was observed in the GPI and GPR groups compared to the control group, with no difference between the GPI and GPR. Conclusion. Under conditions of partial skeletal muscle ischemia, alprostadil did not reduce the release of muscular enzymes, the consumption of tissue glycogen or the effects of ischemia and reperfusion on the cell membrane, characterized by lipid peroxidation.

Effects of the levonorgestrel-releasing intrauterine system on cardiovascular risk markers in patients with endometriosis: a comparative study with the GnRH analogue

Background: The study was conducted to evaluate the cardiovascular risk markers associated with endometriosis and the influence of the levonorgestrel intrauterine system (LNG-TUS) compared with the GnRH analogue (GnRHa) leuprolide acetate on these risk markers after 6 months of treatment. Study Design: This was a randomized, prospective, open clinical Study, with 44 patients with laparoscopically and histologically confirmed endometriosis. Patients were randomized into two groups: the LNG-IUS group, composed of 22 patients who underwent LNG-IUS insertion., and the GnRHa group, composed of 22 patients who received a monthly GnRHa injection for 6 months. Body mass index systolic and diastolic arterial blood pressure; heart rate; and laboratory cardiovascular risk markers such as interlelikin-6 (IL-6), tumor necrosis factor-alpha (TNF-alpha), C-reactive protein (CRP), homocysteine (HMC), lipid profile, total leukocytes and vascular cell adhesion molecule (VCAM) were measured before and 6 months after treatment. Results: After 6 months of treatment, a significant reduction in pain score occurred in both groups with no significant difference in improvement between the two medications evaluated. In the LNG-IUS group, from pretreatment to posttreatment period, there was a significant reduction in the levels (mean +/- SD) of VCAM (92.8 +/- 4.2 to 91.2 +/- 2.7 ng/mL, p=.04), CRP (0.38 +/- 0.30 to 0.28 +/- 0.21 mg/dL, p=.03), total cholesterol (247.0 +/- 85.0 to 180.0 +/- 31.0 mg/dL, p=.0002), triglycerides (118.0 +/- 76.0 to 86.5 +/- 41.5 mg/dL, p=.003), low-density lipoprotein cholesterol (160.5 +/- 66.0 to 114.5 +/- 25.5 mg/dL, p=.0005) and high-density lipoprotein cholesterol (63.0 +/- 20.5 to 48.5 +/- 10.5 mg/dL, p=.002). The GnRHa group showed an increase in HMC levels (11.5 +/- 2.9 to 13.0 +/- 2.7 mu mol/L, p=.04) and a reduction in IL-6 levels (4.3 +/- 3.9 to 2.3 +/- 0.8 pg/mL, p=.005), VCAM (94.0 +/- 3.8 to 92.0 +/- 1.6 ng/mL, p=.03) and total leukocytes (7330 +/- 2554 to 6350 +/- 1778, p=.01). In the GnRH group, the remaining variables, including lipid profile, did not show any statistical difference. Conclusions: This study shows that some cardiovascular risk markers are influenced by both GnRHa and the LNG-TUS, but the latter had a greater positive impact on the lipid profile, which could lead to a favorable effect during long-term treatment. (C) 2010 Elsevier Inc. All rights reserved.

Solid-state NMR structure determination of melittin in a lipid environment

Solid-state C-13 NMR spectroscopy was used to investigate the three-dimensional structure of melittin as lyophilized powder and in ditetradecylphosphatidylcholine (DTPC) membranes. The distance between specifically labeled carbons in analogs [1-C-13]Gly3-[2-C-13]Ala4, [1-C-13]Gly3-[2-C-13]Leu6, [1-C-13]Leu13-[2-C-13]Ala15, [2-C-13]Leu13-[1-C-13]Ala15, and [1-C-13]Leu13-[2-C-13]Leu16 was measured by rotational resonance. As expected, the internuclear distances measured in [1-C-13]Gly3-[2-C-13]Ala4 and [1-C-13]Gly3-[2-C-13]Leu6 were consistent with alpha -helical structure in the N-terminus irrespective of environment. The Internuclear distances measured in [1-C-13]Leu13-[2-C-13]Ala15, [2-C-13]Leu13-[1-C-13]Ala15, and [1-C-13]Leu13-[2-C-13]Leu16 revealed, via molecular modeling, some dependence upon environment for conformation in the region of the bend in helical structure induced by Pro14. A slightly larger interhelical angle between the N- and C-terminal helices was indicated for peptide in dry or hydrated gel state DTPC (139 degrees -145 degrees) than in lyophilized powder (121 degrees -139 degrees) or crystals (129 degrees). The angle, however, is not as great as deduced for melittin in aligned bilayers of DTPC in the liquid-crystalline state (similar to 160 degrees) (R. Smith, F. Separovic, T. J. Milne, A. Whittaker, F. M. Bennett, B. A. Cornell, and A. Makriyannis, 1994, J. Mol, Biol 241:456-466). The study illustrates the utility of rotational resonance in determining local structure within peptide-lipid complexes.

Hydromorphone-3-glucuronide - A more potent neuro-excitant than its structural analogue, morphine-3-glucuronide

In humans, hydromorphone (HMOR) is metabolised principally by conjugation with glucuronic acid to form hydromorphone-3-glucuronide (H3G), a close structural analogue of morphine-3-glucuronide (M3G), the major metabolite of morphine. In a previous study we described the biochemical synthesis of H3G together with a preliminary evaluation of its pharmacology which revealed that it is a neuro-excitant in rats in a manner analogous to M3G. Thus the aims of the current study were to quantify the neuro-excitatory behaviours evoked by intracerebroventricular (icv) H3G in the rat and to define its potency relative to M3G. Groups of adult male Sprague-Dawley rats received icy injections (1 muL) of H3G (1 - 3 mug), M3G (2 - 7 mug) or vehicle via a stainless steel guide cannula that had been implanted stereotaxically seven days prior to drug administration. Behavioural excitation was monitored by scoring fifteen different behaviours (myoclonic jerks, chewing, wet-dog-shakes, rearing, tonic-clonic-convulsions, explosive motor behaviour, grooming, exploring, general activity, eating, staring, ataxia, righting reflex, body posture, touch evoked agitation) immediately prior to icy injection and at the following post-dosing times: 5, 15, 25, 35, 50, 65 and 80 min. H3G produced dose-dependent behavioural excitation in a manner analogous to that reported previously for M3G by our laboratory and reproduced herein. H3G was found to be approximately 2.5-fold more potent than M3G, such that the mean (+/- S.D.) ED50 values were 2.3 (+/- 0.1) mug and 6.1 (+/- 0.6) mug respectively. Thus, our data clearly imply that if H3G crosses the BBB with equivalent efficiency to M3G, then the myoclonus, allodynia and seizures observed in some patients dosed chronically with large systemic doses of HMOR, are almost certainly due to the accumulation of sufficient H3G in the central nervous system, to evoke behavioural excitation. (C) 2001 Elsevier Science Inc. All rights reserved.

Low-temperature single-crystal Raman and neutron-diffraction study of the hydrogenous ammonium copper(II) tutton salt and the deuterated analogue in the metastable state

Low-temperature (15 K) single-crystal neutron-diffraction structures and Raman spectra of the salts (NX4)(2)[CU(OX2)(6)](SO4)(2), where X = H or D, are reported. This study is concerned with the origin of the structural phase change that is known to occur upon deuteration. Data for the deuterated salt were measured in the metastable state, achieved by application of 500 bar of hydrostatic pressure at similar to303 K followed by cooling to 281 K and the subsequent release of pressure. This allows for the direct comparison between the hydrogenous and deuterated salts, in the same modification, at ambient pressure and low temperature. The Raman spectra provide no intimation of any significant change in the intermolecular bonding. Furthermore, structural differences are few, the largest being for the long Cu-O bond, which is 2.2834(5) and 2.2802(4) Angstrom for the hydrogenous and the deuterated salts, respectively. Calorimetric data for the deuterated salt are also presented, providing an estimate of 0.17(2) kJ/mol for the enthalpy difference between the two structural forms at 295.8(5) K. The structural data suggest that substitution of hydrogen for deuterium gives rise to changes in the hydrogen-bonding interactions that result in a slightly reduced force field about the copper(II) center. The small structural differences suggest different relative stabilities for the hydrogenous and deuterated salts, which may be sufficient to stabilize the hydrogenous salt in the anomalous structural form.

Low-voltage low-power CMOS analogue circuits for Gaussian and uniform noise generation

IEEE International Symposium on Circuits and Systems, MAY 25-28, 2003, Bangkok, Thailand. (ISI Web of Science)

Sarcoplasmic reticulum calcium ATPase is inhibited by organic vanadium coordination compounds: pyridine-2,6-dicarboxylatodioxovanadium(V), BMOV, and an amavadine analogue

Inorg Chem. 2008 Jul 7;47(13):5677-84. doi: 10.1021/ic702405d