The PPII-to-α-helix transition of poly-l-lysine in methanol/water solvent mixtures accompanied by fibrillar self-aggregation: An influence of fluphenazine molecules.

Fourier-transform infrared, vibrational circular dichroism spectroscopy and transmission electron microscopy are used to follow the structural changes of pure and fluphenazine (FPh)-mixed poly-l-lysine (PLL) triggered by variations of the methanol to water ratio in solvent mixtures. FPh molecules are used as an effective psychotic drug but with a strong Parkinson's-related side effect. To answer the question whether FPh molecules can modify the fibril development, the PLL polypeptide was used as a model of α-helix- and PPII-rich fibrils. It was stated that the presence of FPh molecules did not inhibit the creation of both types of PLL fibrils with clustering features. The methanol-poor aqueous solutions promote the formation of extended polyproline II (PPII) helices; however, the methanol-rich aqueous solutions induce the development of α-helices of both pure and FPh-mixed PLL. Unpredicted and interesting features of PLL fibrillogenesis are evidenced by the formation of uncommon fibrillar aggregates, which are developed in methanol/water solvents from PLL molecules rich in either α-helix or PPII structures. Possibility of PLL molecules to form ß-sheet-, α-helix- and PPII-rich fibrils demonstrating that fibrillogenesis is a common phenomenon, and fibrillar aggregates can be based on all of the basic protein secondary structures.

[Effect of Macrovipera lebetina and Cerastes cerastes venoms on adherence to integrins of cancerous cells (IGR39, HT29-D4 and IGROV1)]. / Effet des venins de Macrovipera lebetina et de Cerastes cerastes sur l'adherence aux intégrines des cellules cancéreuses (IGR39, HT29-D4 et IGROV1).

In this work, we provide experimental arguments in favor of the fact that components from Macrovipera lebetina and Cerastes cerastes venoms bind to IGR39 melanoma cells but not to HT29D4 cells that derive from carcinoma adenome. Furthermore, Macrovipera lebetina and Cerastes cerastes venoms inhibit the adherence of IGR39 and HT 29-D4 to various extracellular matrix proteins. Macrovipera lebetina and Cerastes cerastes venoms did not inhibit the non specific adherence of IGR 39 cells to polylysine. In addition, binding of components from Cerastes cerastes venom to IGR39 cells is inhibited by GRGDS peptide and by monoclonal antibidy anti-av, while these two components have no effect on the adherence of IGR39 to Macrovipera lebetina venom.

Physicochemical properties and nuclease resistance of antisense-oligodeoxynucleotides entrapped in the core of polyion complex micelles composed of poly(ethylene glycol)-poly(L-lysine) block copolymers.

In this study, the physicochemical properties of polyion complex (PIC) micelles formed from antisense-oligodeoxynucleotides (antisense-ODN) and poly(ethylene glycol)-poly(L-lysine) block copolymers (PEG-PLL) were investigated to utilize them as a novel formulation for antisense-ODN delivery. Angular and concentration dependences of the diffusion coefficient of PIC micelles were evaluated by dynamic light scattering. Results suggested that the formed PIC micelles may have spherical shape with core-shell structure, in which the PIC core formed from antisense-ODN and PLL segment was surrounded by a PEG shell. The average radius of PIC micelles was dependent on the chain length of the PLL segment and was not influenced by the change in the length of ODN molecules at least in the range between 15 and 20 base pairs. Critical association concentration (cac) of PIC micelles was then determined from a profile of light scattering intensity versus concentration (Debye plots). Cac is ca. 0.20 mg/ml, which is low enough to ensure the micelle stability in very diluted condition as is the case with systemic injection into the blood compartment for antisense-ODN therapy. Furthermore, the stability of antisense-ODN against deoxyribonuclease I (DNase I) attack was evaluated using capillary gel electrophoresis, revealing that the complexation of antisense-ODN with PEG-PLL effectively prohibited DNase I attack. These characteristics of the PIC micelle system highlight its promising feature as ODN carrier used in the field of targeting therapy.

Specificity of helix-induction by 2,2,2-trifluoroethanol in polypeptides.

The specificity of helix-induction in polypeptides by 2,2,2-trifluoro ethanol (TFE) is studied using an all beta-sheet protein such as cardiotoxin analogue I (CTX I) from the Taiwan Cobra (Naja naja atra) and a homopolymer such as poly-L-lysine. It is found that alcohols including TFE can 'non-specifically' induce helix at high concentrations both in CTX I and polylysine at neutral pH. However, among the alcohols used, only TFE could transform the heat-induced beta-sheet conformation of polylysine at pH 11.5 into an alpha-helix. The beta-sheet to alpha-helix conversion in polylysine (in the beta-sheet conformation) occurs even at very low concentrations of TFE (< 5% v/v). In addition, experiments on the effect(s) of TFE on the denatured and reduced CTX I (rCTX I) indicate the helix-induction in rCTX I takes place at low TFE concentrations (< 20% v/v). The results of this study hint at the possible influence of disulfide bridges on the induction of helix by TFE.

Acetonitrile-induced conformational transitions in poly-L-lysine.

The effect of acetonitrile on the random coil, alpha-helix and beta-sheet conformations induced in poly-L-lysine is studied. It is found that acetonitrile at higher concentrations transforms the backbone of polylysine from a random coil to a helical conformation. Addition of acetonitrile to polylysine (pH 11.5) in the alpha-helix conformation, induces conformational changes in two stages. At concentrations below 60% v/v, acetonitrile stabilizes the helical conformation and at higher concentrations (> 70% v/v), it destabilizes the helix. beta-sheet-->alpha-helix-->random coil conformational transitions are found to occur when polylysine in the heat-induced conformation is titrated with acetonitrile. The possible mechanism(s) of action of acetonitrile in inducing these structural transitions is discussed.

The transition of alpha-helix to beta-structure of poly(L-lysine) induced by phosphatidic acid vesicles and its kinetics at alkaline pH.

Static and dynamic circular dichroism (CD) measurements were carried out for poly(L-lysine) in suspensions of dilauroylphosphatidic acid (DLPA) vesicles at alkaline pH (8-11.5). The static experiments demonstrated that the alpha-helix of poly(L-lysine) induced by deprotonation in alkaline solutions is transformed to beta-structure by the addition of DLPA vesicles. Stopped-flow CD measurements for such order-to-order transition revealed that the rate determining step is the unfolding process of alpha-helix to random coil. Previously, we have reported the conformational change of poly(L-lysine) induced by DLPA vesicles at neutral pH, where the beta-structure transition from random coil was observed. Thus two types of transition of poly(L-lysine) are observed depending on bulk pH, i.e., from random coil to beta-structure and from alpha-helix to beta-structure. So far the phospholipid-induced conformations of poly(L-lysine) were interpreted in terms of counterbalance between the positively charged terminals of the lysyl chains and the negative headgroups of the phospholipid in vesicle. However, present work indicates the direct interaction other than electrostatic interaction between the lysyl chain and phosphate groups of the lipid.

Structure-selective anesthetic action of steroids: anesthetic potency and effects on lipid and protein.

Alphaxalone was a clinically used steroid anesthetic. Its analog delta 16-alphaxalone is nonanesthetic. The only difference between the two is the presence of a double bond at the hydrophobic end of the delta 16-alphaxalone molecule. This study determined the anesthetic potency of alphaxalone and delta 16-alphaxalone in goldfish and compared it with their effects on dipalmitoylphosphatidylcholine (DPPC) membranes and an alpha-helix polypeptide, poly(L-lysine). The goldfish EC50 values were: alphaxalone 5 mumol/L and delta 16-alphaxalone 80 mumol/L. Because these steroids are insoluble to water, the bulk of the steroid in water is absorbed by the fish. Larger containers hold more steroids than smaller containers at the same steroid concentrations. Then, EC50 values vary according to the size of the container. By assuming that the total amount of steroids in the container is distributed into the fish, the EC50 values expressed by the concentration in the fish body become 1.9 mmol/L for alphaxalone, and 30.5 mmol/L for delta 16-alphaxalone. A monoamino acid peptide, poly(L-lysine), can be formed into random-coil, alpha-helix, or beta-sheet. Addition of 0.07 mmol/L alphaxalone to the alpha-helix poly(L-lysine) partially transformed it to a beta-sheet structure. An equivalent change was observed with 3.0 mmol/L delta 16-alphaxalone. These values translate into 3.5 mmol/L for alphaxalone and 0.15 mol/L for delta 16-alphaxalone, when expressed by the concentration in the peptide. The change from alpha-helix to beta-sheet is accompanied by dehydration of the surface of poly(L-lysine). The steroids decreased the phase-transition temperature of DPPC membrane.(ABSTRACT TRUNCATED AT 250 WORDS)

The modification of proteins by 3-hydroxykynurenine.

The autoxidation and reactivity towards proteins of 3-hydroxykynurenine, a tryptophan metabolite found in the human lens, has been studied. At neutral pH, 3-hydroxykynurenine was readily oxidized using molecular oxygen with the formation of several coloured products. The autoxidation of both 3-hydroxykynurenine and the related aminophenol, 3-hydroxyanthranilic acid, was inhibited by the inclusion of sulphydryl compounds such as glutathione or cysteine. Covalent adducts involving the thiols were not observed with either aminophenol. 3-Hydroxykynurenine was found to react with proteins, including lens proteins, to produce brown-coloured polypeptides characterized by an indistinct long wavelength absorption. This protein tanning was inhibited by glutathione. Despite the presence of an amino group in the side chain of 3-hydroxykynurenine, this tanning of proteins was found to involve amino groups including those of lysine residues, as has been found for 3-hydroxyanthranilic acid. Although both aminophenols tanned polylysine, only 3-hydroxykynurenine induced precipitation of the polyamino acid. 3-Hydroxykynurenine tanned all of the purified crystallins but induced precipitation only in the case of alpha A-crystallin. The implications of these findings for senile cataract are discussed.

Anesthetic-protein interaction: effects of volatile anesthetics on the secondary structure of poly(L-lysine).

Effects of volatile anesthetics (chloroform, halothane, and enflurane) on the secondary structure of poly(L-lysine) were analyzed by circular dichroism (CD). The relative proportions among alpha-helix, beta-sheet, and random-coil conformations were calculated by the curve-fitting method on the CD data. Volatile anesthetics partially transformed alpha-helix to beta-sheet but not to random-coil under the present experimental condition. When expressed by the anesthetic partial pressures in the gas phase in equilibrium with the solution, the values that partially transformed alpha to beta conformation by 10% were 1.1 x 10(-2), 4.7 x 10(-2), and 7.9 x 10(-2) atm for chloroform, halothane, and enflurane, respectively. The order of potency is in reasonable agreement with the order of the anesthetic potencies of the agents. The alpha-to-beta transition was completely reversible when anesthetics were purged by nitrogen gas. Volatile anesthetics disrupted the hydrogen bonds of alpha-helix backbones and rearranged them to form the beta-sheet conformation. The beta-sheet conformation is stabilized mainly by the hydrophobic interaction among methylene side groups of poly(L-lysine). Volatile anesthetics promoted the transition by enhancing the hydrophobic interaction among side-chains and by rearranging the hydrogen bonds in the peptide backbone.